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Taira SATO, Akinori KOCHI, Yuki SHIROSAKI, Satoshi HAYAKAWA, Mamoru AIZAWA, Akiyoshi OSAKA, [Masanori KIKUCHI](https://orcid.org/0000-0002-9451-8147)

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[Preparation of injectable hydroxyapatite/collagen paste using sodium alginate and influence of additives](https://mdr.nims.go.jp/datasets/3d476159-889c-420e-921a-5d5fc46fe123)

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Preparation of injectable hydroxyapatite/collagen paste using sodium alginate and influence of additivesPreparation of injectable hydroxyapatite/collagen pasteusing sodium alginate and influence of additivesTaira SATO,*,** Akinori KOCHI,**,*** Yuki SHIROSAKI,***,‡ Satoshi HAYAKAWA,***Mamoru AIZAWA,* Akiyoshi OSAKA*** and Masanori KIKUCHI**,³*School of Science and Technology, Meiji University, 1–1–1 Higashimita, Tama-ku, Kawasaki 214–8571, Japan**Biomaterials Unit, National Institute for Materials Science, 1–1 Namiki, Tsukuba, Ibaraki 305–0044, Japan***Graduate School of Natural Science and Technology, Okayama University,3–1–1 Tsushima-naka, Kita-ku, Okayama 700–8530, JapanPreparation of injectable hydroxyapatite/collagen bone-like nanocomposite (HAp/Col) paste utilizing sodium alginate (Na-Alg)and influence of additives on the paste properties were investigated. The HAp/Col at the HAp and collagen mass ratio of 80:20synthesized by a simultaneous titration method was pelletized to 100­212¯m. The HAp/Col powder and that treated with100mM CaCl2 (Ca-HAp/Col) were used as starting powders. A paste was prepared by mixing of the starting powder and Na-Algsolution at one of several powder/liquid ratios. The paste was directly injected into 100mM CaCl2 solution to confirm settingcapability, and examined a viscosity, hardening behavior, compressive strength and decay property. In addition, the pastesupplemented with an organic acid or a calcium compound as an additive was also prepared to examine influences of the additiveon paste properties. The organic acid was chosen from citric, succinic, malic, lactic and glycolic acids, and the calcium compoundwas chosen from Ca(OH)2, Ca(CO)3, CaSO4·0.5H2O and calcium citrate.The prepared paste was set by direct injection to 100mM CaCl2 solution. The suitable mixing conditions of the paste were a P/Lratio of 0.6 at the 90:10 mass ratio of Ca-HAp/Col and solution of low viscous Na-Alg. All additives increased the paste viscosity;however, the mechanisms were different between organic acids and calcium compounds. Organic acids rapidly decreased pH toform Alg gel by deposition of Alg. Contrarily, calcium compounds supplied Ca2+ ions to form egg-box structure for gelation ofAlg, and the reaction depended on solubility of compound. Additives also increased decaying time but could not realize anti-decayin the present conditions. The results suggested that the HAp/Col injectable paste can be utilized in rapid prototyping materialsand might be good candidate for injectable artificial bone with further improvement in anti-decay property.©2013 The Ceramic Society of Japan. All rights reserved.Key-words : Hydroxyapatite/collagen bone-like nanocomposite, Injectable paste, Artificial bone, Sodium alginate, Anti-decayproperty, Organic acids, Calcium compounds[Received May 15, 2013; Accepted July 1, 2013]1. IntroductionLarge bone defect, caused by disease or injury, cannot berepaired without using a bone filler. Among bone fillers, autolo-gous bone is still recognized as the best material and used as agold standard for rapid remodeling including bone regenera-tion, because autologous bone contains patient’s own cells andcytokines as well as hydroxyapatite nanocrystals and collagenwith an appropriate nanostructures. Even though, autologous bonetransplantation still has considerable problems relating donor site.The healthy donor site is damaged by secondary surgery, andthe harvesting amount is limited. The donor site remains withoutrecovery to caused long-term pain and the same site is no longeravailable for another surgery. Therefore, the development of betterartificial bone has been strongly desired.Artificial bones composed of bioactive ceramics are widelyused in medical and dental fields in the forms of dense and porousbodies,1),2) glanules3) and pastes.4) Among them, pastes haveadvantages of fluidity to be used in minimally invasive surgeryand formability to fit to the bone defect with complicated shape.The pastes currently used clinically are hydroxyapatite (HAp)-based; one is utilizing reaction of calcium hydrogen phosphatedihydrtate (CaHPO4·2H2O) and tetracalcium phosphate [Ca4-(PO4)2O], and another is utilizing hydration hardening of ¡-tricalicum phosphate [¡-Ca3(PO4)2, ¡-TCP]. Although the crys-tallite size of HAp obtained by these reactions is not so largein comparison to that of sintered HAp, they still require very longperiod to dissolve in our body. A hard HAp block remainspatients’ body for long time to lead severe problems, such as sec-ondary bone fracture and collapsing. Accordingly, preparation ofbiodegradable bone paste is expected from surgeons. A biode-gradable chelate-setting ¢-tricalcium phosphate (¢-TCP) cementusing inositol hexaphosphate was prepared and was absorbed inthe living body.5) This cement showed good properties but stillneeds much improvement on biodegradability. Recently, clinicaltrial data of the hydroxyapatite/collagen nanocomposite (HAp/Col) having similar chemical composition and nanostructure tobone6)­9) conclude that the HAp/Col shows remarkable curativeefficacy rate in comparison to ¢-TCP artificial bone, Osferionμ,which is recognized by surgeons as very efficient artificial bone.10)This result suggested that the HAp/Col paste could be muchbetter material than ¢-TCP cement. To confer hardening andlubrication properties to the HAp/Col, sodium alginate (Na-Alg)³ Corresponding author: M. Kikuchi; E-mail: KIKUCHI.Masanori@nims.go.jp‡ Present affiliation is Frontier Research Academy for YoungResearchers, Kyushu Institute of Technology.Journal of the Ceramic Society of Japan 121 [9] 775-781 2013 Paper©2013 The Ceramic Society of JapanDOI http://dx.doi.org/10.2109/jcersj2.121.775775http://dx.doi.org/10.2109/jcersj2.121.775is the one of the best candidates because it has been already inves-tigated as biomaterials for long time by many researchers,11)­13)and Na-Alg is hardened by chelating to form egg-box structurewith multivalent cations including Ca2+ ion14) which contains inHAp and is known as a good lubricant.15)In the present paper, the HAp/Col paste was prepared bymixing with Na-Alg with and without an additive. Properties ofthe paste obtained were measured by scanning electron micros-copy, compressive strength test, viscosity test, hardening propertytest and decay property test. The optimal conditions for the HAp/Col-alginate paste without additive were determined, and influ-ences of the additive on paste properties were investigated.2. Materials and methods2.1 MaterialsThe HAp/Col with the HAp and collagen mass ratio of 80:20was prepared by a simultaneous titration method.6),16) Briefly, for10 g of the HAp/Col, 100 cm3 of 400mM Ca(OH)2 (preparedfrom alkaline analysis grade CaCO3 (Wako Pure Chemicals Inc.,Japan) suspension and 200 cm3 of 120mM orthophosphoric acid(Reagent grade, Wako Chemicals Inc., Japan) solution with 2.0 gof type-I porcine dermal collagen (Biomaterial Grade, NittaGelatin Inc., Osaka, Japan) solution were simultaneously titratedvia tube pumps to the reaction vessel, in which 100ml of purewater was previously added, with maintaining of temperature at37°C and pH at 9. The HAp/Col obtained was analyzed by X-raydiffraction (XRD, Rigaku, RINT-Ultima III) and thermogravim-etry-differential thermal analysis (TG­DTA, Rigaku, ThermoPlus, Japan). The HAp/Col obtained was compacted at 32mmin diameter, 2mm in height disk using mold specially designedfor squeezing water by uniaxial pressing at 20MPa and freeze-dried, the HAp/Col compacted was then crushed into 100­212¯m in size, and collagen molecules in the HAp/Col powderwere dehydrothermal cross-linked at 140 degrees for 12 h undervacuum. Primary hardening reaction expected for the paste waspartial dissolution of HAp nanocrystals in the HAp/Col to releaseCa2+ ions for gelation of Alg. Thus, presence of Ca2+ ions inliquid phase of the paste was important. However, according toSotome et al.,17) the HAp/Col adsorbed Ca2+ and Mg2+ ions inthe cell culture medium. To inhibit adsorption of Ca2+ ions in theliquid phase of the paste, the HAp/Col powder was stirred in20mM CaCl2 solution for 3 days to be saturated for Ca2+ adsorp-tion, followed by filtering, freeze-drying and re-sieving to collectpowders 100­212¯m in size to obtain Ca-adsorbed HAp/Col(Ca-HAp/Col) powder. The HAp/Col and Ca-HAp/Col pow-ders were observed with scanning electron microscope (SEM,JSM5600LV, JEOL, Japan). Sodium alginates used in the experi-ment were 80­120 (L-Na-Alg), 300­400 (M-Na-Alg), 500­600(H-Na-Alg) cP in viscosity at 10 g/dm3 (Wako Pure ChemicalsInc.) The pastes were prepared by mixing of the Na-Alg solu-tion with HAp/Col or Ca-HAp/Col powder under conditionssummarized in Table 1. To confirm if the amount of Na-Algwas enough, the paste prepared was directly injected into 100mmol/dm3 CaCl2 (Wako Pure Chemicals Inc., Japan) aqueoussolution, this amount is usually enough to form gel by inject-ing Na-Alg solution, using syringe (Thermo, 2mm in caliber).The noodle-like paste was harvested from solution after oneminute soaking and observed their conditions by naked eye andfingers.Gelation of Alg by egg-box formation via Ca2+ would be anessential factor to control setting time and anti-decay propertyof the paste. Release amounts of Ca2+ can be controlled by anorganic acid to dissolve HAp in the HAp/Col or by Ca com-pounds with optimal solubility. Therefore, possible candidates foradditive would be an organic acid or a Ca compound. An organicacid additive was chosen from citric acid (tricarboxylic com-pound), succinic acid, malic acid (dicarboxylic compounds withdifferent carbon chain length), lactic acid and glycolic acid(monocarboxylic compounds with different carbon chain length.)Amounts of organic acid were 0.1, 1 and 10mass% of HAp/Colaccording to the preliminary experiment to determine acceptableamount of lactic acid to maintain injectability of Na-Alg solution.A Ca compound additive was chosen from Ca(CO)3, calciumcitrate (Ca-Cit,) Ca(OH)2 and CaSO4·0.5H2O (ordered in smallto large solubility.) Amounts of Ca compound were 0.5©, 1.0©and 2.0© of equivalent reaction amount (1.67 « 0.07mmol per1 g of Na-Alg) of Ca ion to Na-Alg preliminary measured. Thesechemicals for the additive were purchased from Wako PureChemicals Inc. The additive except for lactic acid, provided asliquid, was added in a powder form. The Ca-HAp/Col paste,which prepared at the Ca-HAp/Col to the L-Na-Alg mass ratio of90:10 and the Ca-HAp/Col powder to the Na-Alg solution massratio of 0.60, was used to investigate influences of an additive.One additive chosen from Ca compounds or an organic acids wasadded to the Ca-HAp/Col paste while mixing.2.2 Viscosity and hardening behavior testsViscosity of the paste prepared was measured according toIshikawa et al.18) Briefly, 0.1 cm3 of the paste was mixed for3min and a 2 kg glass plate was placed on the paste in 10minafter the start of mixing. The spread area was then measured at10min after placing the glass.According to the preliminary test, a hardening behavior testdescribed in the Japanese industrial standard JIS T 6602 fordental zinc phosphate cement could not be applied for the HAp/Col and Ca-HAp/Col pastes because of softness of the pastes.Thus, a hardening behavior of the paste was investigated by time-dependent viscosity test. The 0.3 cm3 samples, to distinguish achange of value, were packed in silicone tube mold, and wereincubated for 1, 2, 3 and 4 days in an incubator maintained itstemperature at 37°C and relative humidity of 100%. The pastestested were prepared under conditions summarized in Table 2.In addition, the influence of additive on initial hardeningbehavior was measured using Ca-HAp/Col paste with 2.0© Ca-Cit and that with 10mass% succinic acid to Ca-HAp/Col pow-der at 1, 6 and 24 h after incubation by the viscosity test.Table 1. Concentration of Na-Alg solution at each conditions(HAp/Col)/Na-Algweight ratioP/L ratio0.82 0.69 0.60 0.53 0.47 0.4395/5 4.8 4.0 3.5 3.0 2.7 2.490/10 9.1 7.7 6.7 5.9 5.3 4.880/20 16.7 14.3 12.5 11.1 10.0 9.1Table 2. Powder/liquid ratios for hardening behavior and compressivestrength testsMolecular weight of Na-AlgMass ratio of HAp/Col to Na-Alg90:10Low 0.60*Middle 0.53High 0.53*for hardening behaviorSato et al.: Preparation of injectable hydroxyapatite/collagen paste using sodium alginate and influence of additivesJCS-Japan7762.3 Compressive strength testThe samples were prepared according to Table 2. The pastewas packed in a silicon tube mold (5mm inside diameter, 6mmin height), placed in an incubator for 1, 2, 3 and 4 days. Thecompressive strength of the paste was then measured witha texture analyzer (TA-XT2i, Stable Micro Systems Inc.) withplunger of 10mm diameter and head speed of 1mm/min.2.4 Decay property testDecay property for the paste was measured by the procedurein Japanese industrial standard JIS T 0330-4 Bioceramics-Part4:Characterization of calcium phosphate paste. In detail, after 3minmixing of raw materials, the paste mixed was packed in thesyringe of 4.8mm in inner diameter and 16.5mm in height.Within 5min after the start of mixing, the paste was squeezed onwire net with wire diameter of 0.5mm and aperture of 2.0mm,and was soaked into 50 cm3 of 37°C phosphate buffered saline(PBS). The paste in PBS was then statically placed at 37°C for72 h in an incubator. Decay rate was calculated from the finalweight of paste left on the net, and decaying time for the pastethat completely decayed within 72 h was also measured.3. Results and discussionFrom a result of the TG­DTA measurement, mass ratio ofinorganic phases to total mass without adsorbed water for theHAp/Col prepared was 80.2% and was almost the same asstarting material ratio.Powder X-ray diffraction pattern of the as-prepared HAp/Colshowed the low-crystalline HAp, and that of the HAp/Col afterheating at 1200°C showed presence of 16.7mass% ¢-TCP;therefore, the inorganic phase of HAp/Col obtained was assumedto be a Ca deficient carbonate containing HAp, Ca9.8(CO3)0.4-(PO4)5.6(OH)2.As shown in Fig. 1, no significant differences of particlemorphology between HAp/Col and Ca-HAp/Col powders wereobserved with SEM images as well as specific surface areas ofthem, 55.1 « 3.7m2·g¹1 for the HAp/Col and 58.2 « 3.8m2·g¹1for the Ca-HAp/Col powders. Accordingly, adsorption treatmentof Ca2+ ion on the HAp/Col did not affect apparent conditions ofHAp/Col powder.The paste poured into CaCl2 aqueous solution hardenedimmediately as shown in Fig. 2(a), and that collected from thesolution had enough strength to handle in surgical and cellculture operation with viscoelasticity as shown in Fig. 2(b); thus,10mass% of Alg in the paste was enough to cross-link viaCa2+ ions. In addition, this property is expected to apply the pasteto rapid prototyping system for preparing fine controlled tissueengineering scaffolds.Figure 3 shows results of viscosity test for various conditions.Viscosity increased (spread area decreased) with increasing inP/L ratio and/or increasing in viscosity of Na-Alg used. Themost suitable P/L ratio of each pastes are determined as Table 3,the conditions that spread area revealed 200mm2, because thepaste had good unity and had sufficient but not superabundantwater content to allow injection.Figures 4 and 5 shows the results of viscosity test for the pasteprepared with an organic acid and a Ca compound, respectively.Effect of additive on viscosity depended on its amount. For theorganic acid added paste, number of carboxy group(s), size ofmolecules and/or chelate ability in organic acid had no influenceson viscosity; thus, increase of paste viscosity could be only theinfluence of acid pH.15) In the case of Ca compounds addi-tion, the viscosity of the paste increased with increasing in boththe amount and solubility of the Ca compounds. These resultssuggested that viscosity of the paste increased by the cross-link ofNa-Alg in the paste by dissolved Ca2+ ions from the additive.At the mass ratio of HAp/Col or Ca-HAp/Col to Na-Alg 95:5, the paste prepared was very fragile at 1 day after incubationdue to insufficiency of Na-Alg amount to fix the paste. Other Na-Alg ratio seemed to be good; however, to maximize the HAp/ColFig. 1. SEM image of (a) HAp/Col and (b) Ca-HAp/Col.Fig. 2. (a) Ca-HAp/Col paste just after injected into 100mM CaCl2 solution. (b) Ca-HAp/Col paste after immersed 100100mM CaCl2 solution for 1min.Journal of the Ceramic Society of Japan 121 [9] 775-781 2013 JCS-Japan777properties, we concluded that 10mass% of Na-Alg would bemuch better than 20mass% of Na-Alg. The time-departmentchanges in viscosity of the HAp/Col and Ca-HAp/Col pastes atNa-Alg ratio of 10mass% are shown in Fig. 6. Viscosities ofboth pastes increased with time until day 2; however, that of theHAp/Col paste reached plateau at day 3 even that of the Ca-HAp/Col paste was continuously increasing at least till day 4.Fig. 3. Spread area, as an index of consistency, of the Hap/Col and Ca-Hap/Col paste prepared by under various conditions.Mass ratio of Hap/Col or Ca-Hap/Col to Na-Alg was fixed at 95/5, (a) Hap/Col and (b) Ca-Hap/Col paste. Mass ratio ofHap/Col or Ca-Hap/Col to Na-Alg was fixed at 90/10, (c) Hap/Col and (d) Ca-Hap/Col paste. Mass ratio of Hap/Col orCa-Hap/Col to Na-Alg was foxed at 80/20, (e) Hap/Col and (f ) Ca-Hap/Col paste.Table 3. Optimal P/L ratio for each conditionsMolecular weight of Na-AlgMass ratio of HAp/Col to Na-Alg95:5 90:10 80:20Low 0.69 0.60 0.53Middle 0.60 0.53 0.47High 0.60 0.53 0.47Sato et al.: Preparation of injectable hydroxyapatite/collagen paste using sodium alginate and influence of additivesJCS-Japan778These differences could be caused by difference in adsorptionbehavior of Alg on the HAp/Col and Ca-HAp/Col. Amountsof adsorption site, Ca, on the Ca-HAp/Col particles could begreater than those of HAp/Col, because of Ca2+ ion adsorptiontreatment. Initial stage of mixing, large amounts of Alg wereadsorbed on the Ca-HAp/Col particles, subsequently amounts offree Alg for gelation decreased from liquid phase of the Ca-HAp/Col paste. In the meantime, surface of HAp nanocrystals in bothpaste started dissolution by chelating effect of Alg. Some of Algmolecules chelating Ca2+ ion(s) started to cross-link via egg-boxstructures. At this time, lower amounts of free Alg molecules inliquid phase of the Ca-HAp/Col paste allowed formation ofsmaller amounts of cross-links in comparison to the HAp/Colpaste. As a result, viscosity of the Ca-HAp/Col paste was alwayssmaller than that of the HAp/Col paste by day 2. Furthermore,Alg molecules easily cross-linked at neighborhood of the HAp/Col particles due to existence of large amount of Alg moleculesand could inhibit long range Alg gel network formation in theHAp/Col paste. Contrarily, gradual release of Alg moleculeswith Ca2+ ions from Ca-HAp/Col particle surfaces could formlong-range network initially and form much harder gel in com-parison to the HAp/Col paste with formation of stronger networkat 3 or more days after mixing.Figure 7 shows the hardening behavior as a function of timefor the paste prepared with 10mass% succinic acid or 2© Ca-Cit.As a trend, viscosity of the pastes increased with time; however,the manner of hardening was very different between the pasteswith succinic acid and Ca-Cit. With addition of succinic acid, thepaste pH would decrease rapidly to introduce Alg gel formationby deposition of Alg from the Alg solution, then the pasteviscosity decreased to approximately 60mm2 in spread are justafter mixing and reached plateau, approximately 40mm2 inspread area in 6 h. Contrarily, viscosity of the paste prepared withCa-Cit increased gently by slow release of Ca2+ ions from Ca-Fig. 4. Spread area of organic acid added Ca-HAp/Col paste.Fig. 5. Spread area of calcium compound added Ca-HAp/Col paste.Fig. 7. Hardening behavior of additive-added paste.Fig. 6. Spread area of the HAp/col and Ca-HAp/Col pastes prepared atoptimum P/L ratio as a function of incubation time. Mass ratio of HAp/Col or Ca-HAp/Col to Na-Alg was set at 90/10.Journal of the Ceramic Society of Japan 121 [9] 775-781 2013 JCS-Japan779Cit; therefore, initial spread area was the same as that of the non-additive paste. Even though, the paste with Ca-Cit decreased itsviscosity faster than that of the non-additive paste.Figure 8 shows the results of compressive strength test.Compressive strengths of all pastes increased with time withsimilar behavior except for the Ca-HAp/Col paste prepared withL-Na-Alg. The Ca-HAp/Col paste prepared with L-Na-Algonly showed drastic increase of the compressive strength at3 days after incubation than others. This phenomenon could becaused by differences in dispersion rates of Alg molecules in thepastes due to size of Alg molecules in combination with the usageof Ca-HAp/Col powder as described above.On the decay property test, all pastes without the additivedecayed completely in the D-MEM in less than 2 h, and all pasteswith the additive decayed within 24­48 h. Figure 9 shows decaytime of the paste with Ca compound as the additive. Evenviscosity of the paste prepared with CaSO4·0.5H2O or Ca(OH)2increased greater than that with other Ca compounds, no sig-nificant differences in decay time were observed between thepaste with and without additives. Except for them, decay timeshowed the same trend to the viscosity change as well as Ca com-pound solubility. Fundamentally, Ca2+ release from the additivesallowed to form Alg network faster than that released from HAp/Col; thus, viscosity of the paste increased with Ca2+ release rate,solubility. Contrarily, rapid Ca2+ release formed strong Alg gelsurrounding of the Ca compound particles, and it inhibiteddispersion of Ca2+ ions whole through the paste. Accordingly,Alg coagulates with Ca compound core formed in the pasteto increase viscosity but to decrease unity of the paste.In the present conditions, the paste composed of HAp/Col andAlg with non-decay property for an injectable artificial bonecould not obtained; however, combination of organic acid andCa compound would make the injectable artificial bone possibleby control both initial viscosity to resist flow to accelerate decayand faster hardening via strong cross-link of Alg molecules viaCa2+ ions.4. ConclusionThe optimal preparation conditions of the HAp/Col paste arethat the P/L ratio is 0.6 with the 90:10 mass ratio of HAp/Colpowder treated with CaCl2 and low viscous Na-Alg. The pasteprepared formed viscoelastic solid by direct injection into CaCl2aqueous solution. As an additive, organic acid increased pasteviscosity very rapidly and Ca compound accelerate formationof egg-box cross-link in comparison to the non-additive paste.The pastes before hardened were decayed by soaking in D-MEMor PBS, some additive increased time to completely decay.In conclusion, the HAp/Col based paste prepared in the paperwould be applied in rapid prototyping with CaCl2 aqueoussolution soaking and could be a good candidate for injectableartificial bone with a property to completely incorporate intobone remodeling process after improvement of anti-decay prop-erty by using combination of organic acid and Ca compound.Acknowledgement This work was supported in part by JSTfunding for science exchange among Japan, China and Korea.References1) A. Okazaki, T. Koshino, T. Saito and T. Takagi, Biomaterials,21, 483­487 (2000).2) U. Ftipamonti, Biomaterials, 17, 31­35 (1996).3) A. M. Gatti, D. Zaffe and G. P. Poli, Biomaterials, 11, 513­517(1990).4) H. Monma and T. Kanazawa, Yogyo Kyokai Shi, 84, 209­213(1976).5) S. Takahashi, T. Konishi, M. Nishiyama, M. Mizumoto, M.Honda, Y. Horiguchi, K. Oribe and M. Mamoru, J. Ceram. Soc.Japan, 119, 35­42 (2011).6) M. Kikuchi, S. Itoh, S. Ichinose, K. Shinomiya and J. Tanaka,Biomaterials, 22, 1705­1711 (2001).7) S. Itoh, M. Kikuchi, Y. Koyama, K. Takakuda, K. Shinomiyaand J. Tanaka, Biomaterials, 23, 3919­3926 (2002).8) S. Yunoki, T. Ikoma, A. Monkawa, K. Ohta, M. Kikuchi, S.Sotome, K. Shinomiya and J. Tanaka, Mater. Lett., 60, 999­1002 (2006).9) M. Kikuchi, S. Itoh, H. N. Matsumoto, Y. Koyama, K.Takakuda, K. Shinomiya and J. Tanaka, Key EngineeringMaterials, 240–242, 567­570 (2003).10) K. Shinomiya, M. Ishizuki, H. Morioka, S. Matasumoto, T.Nakamura, S. Abe and Y. Beppu, Seikei Geka, 63, 921­926(2012) [in Japanese].Fig. 8. Changes in compressive strength of the HAp/Col and Ca-HAp/Col pastes prepared at optimum P/L ratio as a function of incubationtime. Mass ratio of HAp/Col or Ca-HAp/Col to Na-Alg was set at 90/10.Fig. 9. Decay time of calcium compound added Ca-HAp/Col paste.Sato et al.: Preparation of injectable hydroxyapatite/collagen paste using sodium alginate and influence of additivesJCS-Japan78011) K. Ishikawa, Y. Miyamoto, M. Kon, M. Nagayama and K.Asaoka, Biomaterials, 16, 527­532 (1995).12) S. M. Zhang, F. Z. Cui, S. S. Liao, Y. Zhu and L. Han, Jounalof Materials Science; Materials in Medicine, 14, 641­645(2003).13) L. Wang, R. M. Shelton, P. R. Cooper, M. Lawson, J. T. Triffittand J. E. Barralet, Biomaterials, 24, 3475­3481 (2003).14) E. Stodolak, C. Paluszkiewicz, M. Bogun and M. Blazewicz,J. Mol. Struct., 924–926, 208­213 (2009).15) K. Y. Lee and D. J. Mooney, Prog. Polym. Sci., 37, 106­126(2012).16) M. Kikuchi, T. Ikoma, S. Itoh, H. N. Matsumoto, Y. Koyama,K. Takakuda, K. Shinomiya and J. Tanaka, Compos. Sci.Technol., 64, 819­825 (2004).17) S. Sotome, T. Uemura, M. Kikuchi, S. Itoh, J. Tanaka, M.Takahashi, T. Tateishi and K. Shinomiya, Key Eng. Mater.,218–220, 153­156 (2002).18) K. Ishikawa, Y. Miyamoto, M. Takechi, T. Toh, M. Kon, M.Nagayama and K. Asaoka, J. Biomed. Mater. Res., 36, 393­399 (1997).Journal of the Ceramic Society of Japan 121 [9] 775-781 2013 JCS-Japan781