# Fileset

[Journal of the Ceramic Society of Japan 125 [7] 579-583 2017.pdf](https://mdr.nims.go.jp/filesets/886132c3-c573-41e6-99ef-89ec0935dd3c/download)

## Creator

Naga Vijaya Lakshmi MANCHINASETTY, Taira SATO, Mamoru AIZAWA, Sridharan MADANAGURUSAMY, [Masanori KIKUCHI](https://orcid.org/0000-0002-9451-8147)

## Rights

[Creative Commons BY Attribution 4.0 International](https://creativecommons.org/licenses/by/4.0/)

## Other metadata

[Influences of combined supplementation of calcium citrate and calcium carbonate on injectable and anti-washout hydroxyapatite/collagen bone paste utilizing sodium alginate](https://mdr.nims.go.jp/datasets/b55d3de9-b496-4d06-99a3-5805d3b87238)

## Fulltext

Influences of combined supplementation of calcium citrate and calcium carbonate on injectable and anti-washout hydroxyapatite/collagen bone paste utilizing sodium alginateInfluences of combined supplementation of calcium citrate andcalcium carbonate on injectable and anti-washouthydroxyapatite/collagen bone paste utilizing sodium alginateNaga Vijaya Lakshmi MANCHINASETTY*,**, Taira SATO**,***, Mamoru AIZAWA***,Sridharan MADANAGURUSAMY**** and Masanori KIKUCHI*,**,³*Division of Bioengineering and Bioinformatics, Graduate School of Information Science and Technology,Hokkaido University, Kita-14, Nishi-9, Kita-Ku, Sapporo 060–0814, Japan**Bioceramics Group, Research center for Functional Materials, National Institute for Materials Science,1–1 Namiki, Tsukuba, Ibaraki 305–0044, Japan***Department of Applied Chemistry, Graduate School of Science and Technology, Meiji University,1–1–1 Higashimita, Tama-ku, Kawasaki 214–8571, Japan****Functional Nanomaterials & Devices Lab, Center for Nanotechnology and Advanced Biomaterials, SASTRA University,Thanjavur, Tamil Nadu 613–401, IndiaInfluences of combined supplementation of calcium citrate (Ca­Cit) and calcium carbonate (CaCO3) on the hydroxyapatite/collagen (HAp/Col)­sodium alginate (Na­Alg) paste (HAp/Col paste) were investigated. Supplementation amounts of Ca­Cit andCaCO3 were denoted as Nx, where N is a multiplication number to Ca2+ ion amounts for the equivalent reaction with Na­Algobtained previously. Combined supplementation of 10x Ca­Cit and 2x CaCO3 improved the anti-washout property at a washoutratio of 2.42 « 0.72% and pH maintenance at 7.34 « 0.08, these values were better than the best HAp/Col paste using Ca­Cit(5.91 « 2.73% washout ratio and 6.72 « 0.06 of pH). This is due to coordinate effects of initial washout inhibition by weak butrapid formation of long-range network by citric acid followed by long term anti-washout inhibition by strong but slow networkformation by Ca2+ ions. The paste also showed good cytocompatibilty that MG63 cells proliferated with the culture time withoutany significant difference with the HAp/Col dense bodies. The HAp/Col paste is expected to be utilized in minimally invasivesurgery of bone defect.©2017 The Ceramic Society of Japan. All rights reserved.Key-words : Composites, Biomedical applications, Hydroxyapatite/collagen composite, Injectable bone paste, Calcium citrate,Sodium alginate[Received January 23, 2017; Accepted April 11, 2017]1. IntroductionAutologous bone grafts are considered as the best by surgeons,even though they still face problems, e.g., donor site morbidity,deformity, scarring and chronic pain.1) Artificial bone void fillersbased on bioactive ceramics are widely used and developedto replace or reduce the usage of autologous bones. Recently,clinical use of injectable bone pastes is increasing rapidly dueto their advantages, ease to fit irregular-shaped defects withoutunexpected gaps between host and fillers,2),3) injectability to suitfor minimally invasive surgery, which reduce patient discomfortand complications along with a decrease in health care costs. Themost clinically successful pastes is so-called “apatite cement”that utilize the hydration hardening reaction by conversion ofcomparatively unstable calcium phosphates to hydroxyapatite[Ca10(PO4)6(OH)2, Hap] crystals.4) However, HAp remains inthe patients’ body due to its very low biodegradability, and itsbrittleness possibly lead a secondary bone fracture. In the sinteredHAp, high porosity can solve the problems in part; however, it isdifficult to apply to apatite cements, because addition of suffi-cient porogen decreases the operability and cause low fracturestrength.Application of biodegradable materials is the other solution forbone paste. Biodegradable bone void fillers clinically used are,e.g., porous ¢-tricalcium phosphate [¢-Ca3(PO4)2, ¢-TCP] andhydroxyapatite/collagen bone-like nanocomposite (HAp/Col).Sintered ¢-TCP is clinically used since 1970’s due to it’s poten-tial of dissolution by body fluid and resorption by osteoclasts,which are the main reactions necessary to substitute with newbone. Thus, fabrication of chelate setting ¡- and ¢-TCP pastesusing inositol-6-phosphate as a chelating agent and their potentialin substitution with new bone have been reported.5),6) On theother hand, the HAp/Col was prepared by the simultaneoustitration method and has similar bone like nanostructure, wherec-axes of hydroxyapatite (HAp) nanocrystals were aligned alongcollagen fibrils. It is incorporated into bone remodeling process tosubstitute with newly formed bone when implanted into bonedefect.7),8) Clinical trials on the porous HAp/Col proved to be abetter material than porous ¢-TCP.9) An injectable HAp/Colpaste had been developed using sodium alginate,10) which isalready recognized as a biocompatible material with gelationability by multivalent cations and a good lubricant;11) however,³ Corresponding author: M. Kikuchi; E-mail: KIKUCHI.Masanori@nims.go.jp‡ Publication of this international collaborative article is supportedby JSPS Grants-in-Aid for Scientific Research (KAKENHI), GrantNumber 252016Journal of the Ceramic Society of Japan 125 [7] 579-583 2017 Full paper©2017 The Ceramic Society of JapanDOI http://doi.org/10.2109/jcersj2.17016579http://doi.org/10.2109/jcersj2.17016its anti-washout property was not sufficient for clinical use. Toimprove the anti-washout property, supplementation of variouscalcium compounds or organic acids were investigated;12) how-ever, only slight improvement was achieved by using low solublecalcium compounds, calcium carbonate (CaCO3) and calciumcitrate (Ca­Cit).Recently Sato et al.13) reported influences of excess supple-mentation of low soluble CaCO3 or Ca­Cit for the improvementof anti-washout property. The HAp/Col pastes showed a washoutratio at less than 10% in mass by addition of Ca­Cit at 8 timesor greater Ca2+ ion amounts than that for the equivalent reactionto Na­Alg with an observation of a slight decrease in the pH ofthe PBS after the anti-washout test. Further, a degradation ofthe paste prepared with supplementation of Ca­Cit at 20 timesgreater Ca2+ ion amounts than that for the equivalent reaction toNa­Alg was observed in Dulbecco’s modified essential mediumin 5 days. This result suggests that the formation of acid-inducedalginate gel with high H+ concentration inhibited eggbox struc-ture formation. This phenomenon could be palliated by combinedsupplementation of acidic Ca­Cit and basic CaCO3. In the pres-ent paper, influences of combined supplementation of Ca­Cit andCaCO3 on injectable HAp/Col paste were investigated.2. Materials and methods2.1 Preparation of HAp/Col powderThe HAp/Col at a HAp and collagen mass ratio of 4:1 wasprepared by a simultaneous titration of Ca(OH)2 (prepared fromthe alkaline analysis grade CaCO3, Wako pure chemicals Inc.)suspension and mixed solution of orthophosphoric acid (Reagentgrade, Wako chemicals Inc.) and type-1 porcine dermal collagen(Biomaterial grade, Nitta Gelatin Inc.) with maintaining the waterbath temperature at 40°C and pH of the reaction solution at 9.8)The HAp/Col obtained was compacted into disks by squeezingwater using a uniaxial press at 20MPa, freeze dried and crushedinto 100­212¯m in size with a ball mill (FRITSCH, Pulverisette,Germany) in vacuo using zirconia ball of 15mm¯. The powderobtained was then dehydrothermally crossed linked at 140°C for12 h under a vacuum. In order to inhibit Ca2+ adsorption of theHAp/Col,14) the powder was treated with 20mM CaCl2 solutionfor 3 days, filtered, freeze dried and stored at 4°C.2.2 Characterization of HAp/Col powderInorganic phase of the HAp/Col powder was identified by thepowder X-ray diffractometry (XRD, Rigaku, RINT-Ultima III)using CuK¡ radiation from 2 to 60° of diffraction angle, 2ª, at ascanning rate of 2°/min. Inorganic and organic ratio of the HAp/Col powder was determined by the thermogravimetry-differentialthermal analysis (TG­DTA, Rigaku, ThermoPlus, Japan) fromroom temperature to 1200°C at a heating rate of 5 °C/min. Afterthe TG­DTA analysis, powder was identified by powder XRDand estimated Ca/P atomic ratio by calculation using relativeintensity of 202 diffraction line of HAp (34.06°) and 220diffraction line of TCP (34.34°).2.3 Preparation of HAp/Col pasteThe HAp/Col paste was prepared using the optimal conditionsin Ref. 12, where the powder to liquid (P/L) ratio is 0.60 andmass ratio of HAp/Col powder and sodium alginate [Na­Alg,low viscosity (80­120 cP)], Wako pure chemicals, Inc.) is 9:1.The calcium compound additives chosen were CaCO3 and Ca­Cit (Wako pure chemicals, Inc.) The reaction equivalent amountof Ca2+ ion to Na­Alg used in the experiments was 1.67mmolper 1 g of Na­Alg reported in the Ref. 12. According to this,amounts of supplement was denoted as Nx, where N was multi-plication number for the equivalent reaction amount of Ca2+ ionto Na­Alg. The HAp/Col paste was prepared by a mixing of theHAp/Col and Na­Alg aqueous solution with a combined supple-mentation of Ca­Cit and CaCO3 under the conditions shown inTable 1, in which their abbreviations are also noted.2.4 Viscosity testViscosity of the paste was measured using the method reportedby Ishikawa et al.,15) where 0.1 cm3 of the paste was mixed for3min and a 2-kg glass plate was placed on the paste for 10minafter start of mixing. The spread area at 10min after placing theglass was measured using its digital photograph with the Image-Jprogram (version 1.48, NIH, USA).2.5 Washout property testWashout property of the HAp/Col paste was measured accord-ing to the procedure in Japanese Industrial Standard “JIS T 0330-4 Bioceramics- Part 4”. The paste was prepared by mixing theraw materials for 3min and was packed into a syringe of 4.8mmin inner diameter and 16.5mm in height. Within 5min aftermixing the paste was then squeezed onto a wire net with wirediameter of 0.5mm and aperture of 2.0mm. The paste was thensoaked in 50ml of phosphate buffered saline (PBS) and kept at37°C in an incubator up to 72 h. Awashout rate was calculated asthe ratio of the paste on the wire net before and after soaking. Thefinal pH of the solution was also measured.2.6 Cytocompatibilty testAll paste materials were sterilized using ethylene oxide gas(EOG). The paste raw materials were mixed, molded into theshape of a cylinder of 7mm diameter and 5mm in height andwere aged for 24 h in an incubator at 95 « 5% RH to harden. Ablank and HAp/Col dense body were chosen as controls. HAp/Col dense bodies was prepared by compact dehydration of theHAp/Col as synthesized into 5mm in height and punched outinto 7mm diameter. They were freeze dried, dehydrothermallycrosslinked and sterilized using EOG respectively. Before cellculture experiment, the HAp/Col dense bodies were soaked inTable 1. Preparation conditions of the HAp/col paste with additivesHAp/Col(mg)Na­Alg(mg)H2O(¯l)Ca­Cit CaCO3AbbreviationFinal P/LratioTimesequivalentWeight(mg)TimesequivalentWeight(mg)170 18.9 264.48x 47.9 2x 6.30 8x-2x 0.799x 53.9 1x 3.20 9x-1x 0.8010x 59.9 1x 3.20 10x-1x 0.8210x 59.9 2x 6.30 10x-2x 0.8312x 71.9 2x 6.30 12x-2x 0.87x = Reaction equivalent amount of Ca2+ to Na­Alg (1.67 « 0.07mmol per 1 g of Na­Alg).Manchinasetty et al.: Influences of combined supplementation of calcium citrate and calcium carbonate on injectable and anti-washouthydroxyapatite/collagen bone paste utilizing sodium alginateJCS-Japan580Dulbecco’s Modified Eagle Medium (D-MEM) to adsorb Ca2+ion and Mg2+ ions. MG-63 cells, derived from human osteo-sarcoma was used for the experiment. After a subculture ofMG63 cells, 2 © 104 cells were seeded onto each well in a 6 welltissue culture treated polystyrene plate (TCPS). One day afterseeding, the paste and control samples were placed in each welland cultured for 7 days. Medium was changed every 2 days.After desired time interval, the cells were detached by trypsin/EDTA and the cell numbers were calculated using hemocytom-eter. The used media was collected during the medium change tomeasure the Ca2+ and PO43¹ ion concentrations using an induc-tively coupled plasma-atomic emission spectrometer (SPS7800,SII NanoTechnology, Japan; ICP-AES).2.7 Statistical analysisThe results are shown as the mean « standard deviation. Statis-tical analysis was performed using one way analysis of variancewith Turkey-HSD post hoc test. The statistical significance wasset at p < 0.05.3. Results and discussionThe mass ratio of inorganic phase to total mass of the driedHAp/Col calculated from TG­DTA measurement was 80.2%,which was similar to the ratio of starting materials as described inthe previous report.8) Figure 1 shows the XRD patterns of asprepared HAp/Col and heated treated HAp/Col. Powder X-raydiffraction pattern of the as-prepared HAp/Col showed that theinorganic phase of the HAp/Col was low crystalline HAp. A partof the low crystalline HAp decomposed to ¢-tricalcium phos-phate [¢-Ca3(PO4)2, TCP] by heating at 1200°C, i.e., the lowcrystalline HAp was calcium deficient HAp. Mass ratios ofcrystalline phases of heat treated HAp/Col quantified from theXRD peaks was 84.5% of HAp and 15.5% of TCP, and a Ca/Patomic ratio of the inorganic phase of HAp/Col was 1.63.Spread area of the paste combination supplemented with Ca­Cit and CaCO3 increased with Ca2+ amounts as shown in Fig. 2.Further, spread areas of the pastes supplemented with totally 10xCa2+ ion, approximately 120­130mm2, was very similar to thatof the paste solely supplemented with 10x Ca­Cit as reported bySato et al.13) These results suggested pastes supplemented withcalcium compounds had higher influences on viscosity. Effectson initial viscosity with acid compounds as supplements werelow in comparison to those of Ca2+ amounts. This could becaused by difference between strong eggbox gel and weak acid-induced gel. Anti-washout property became better with increas-ing in total Ca2+ amounts up to 10x-2x and again worse at 12x-2xas shown in Fig. 3. The anti-washout ratios for the paste withFig. 1. XRD patterns of as prepared HAp/Col and the HAp/Col afterheated at 1200°C.Fig. 2. Spread area of the HAp/Col paste combination supplementedwith Ca­Cit and CaCO3. Data represent mean « standard deviation (SD)for n = 3.Fig. 3. Washout behavior and final pH of the medium after 72 h. Datarepresent mean «SD for n = 3.Journal of the Ceramic Society of Japan 125 [7] 579-583 2017 JCS-Japan581combined supplementation demonstrated a smaller value at anamount of 10x-2x (2.42 « 0.72%) in comparison to the 10x ofCa­Cit solely supplemented pastes (5.91 « 2.73%).12) Further,the final pH of the solution was maintained as the original pH forthe 10x-2x (7.34 « 0.08) compared with the 10x of Ca­Citsupplemented paste (6.72 « 0.06);12) though final pH of 10x-1xand 9x-1x increased.In our previous reports,12),13) to improve the anti-washout prop-erty of HAp/Col paste, the paste was supplemented with organicacid or calcium compounds, resulting in increase of viscosityand slight extension of washout time. The mechanisms for theincrease in viscosity were different in each supplement; i.e., acidsincreased viscosity of alginate by acidic environment and calciumcompounds by crosslinkage of alginate via Ca2+ ions. The pastesprepared with high soluble calcium compound showed noimprovements in anti-washout property, because high solublecalcium compounds discharge Ca2+ ions quickly to form shortrange gel near the calcium compounds, which increased in pasteviscosity and inhibits the diffusion of Ca2+ ions to form long-range network to prevent the washout. Contrarily, the pastes pre-pared with low soluble calcium compound, CaCO3 and Ca­Citimproved the anti-washout property. However, paste supple-mented with 20x CaCO3 decayed completely within 72 h anddid not acquire sufficient wash out property. Large amounts ofCaCO3 allowed formation of eggbox gel to increase viscosity;however, the gel formation by low Ca2+ concentration due to lowsolubility of CaCO3, might be insufficient for the anti-washout inPBS by substitution of Ca2+ to Na+, which leads to the decom-position of alginate gel network. Contrarily, Ca­Cit supplemen-tation showed anti-washout property because of alginate gelationby acidic pH. Acidic alginate gel might be stronger than the gelformed by small amounts of Ca2+ under PBS condition. In addi-tion, Ca­Cit in the paste also formed the egg-box structuregradually by large amount of Ca2+ to reinforce the paste. How-ever, the pH of the PBS after the anti-washout test became acidic,which can compromise the biocompatibility of the paste.Similar reactions occurred in the paste with combined supple-mentation of Ca­Cit and CaCO3. First, Ca­Cit, comparativelyhigher solubility than CaCO3, dissolved in the paste and allowedto form weak acid-induced gel. Dissolution of CaCO3 wasfollowed by acid environment and increased the pH, thus weak-ening the acid-induced gel. In the meantime, Ca2+ ions startedto interact with gelation sites of alginate due to increase of freeCa2+ ions from chelation with citrates; this meant that the reasonof small improvement in the wash out property by the solelysupplementation of Ca­Cit12) could not only be by inhibition ofthe eggbox formation by acid-induced gel but also by inhibitionof interaction between Ca2+ ions and the eggbox sites. This is dueto competitive reaction between the chelation by citrate and theeggbox formation.Supply of Ca2+ ions from CaCO3 less than or equal to 10%, theamount of Ca2+ ions might not be sufficient for moving reactionequivalent to the eggbox structure formation; thus, the free Ca2+ions leached from the paste and increased the pH. Contrarily, Ca2+ions supplied from CaCO3 more than 10%, were used for theformation of eggbox structure as well as chelating with citrates;therefore, pHs were comparatively neutral than small Ca2+ ionssupplied from CaCO3. Further, initial acid-induced gel formationwas also important for anti-washout property; thus, the 10x-2xpaste showed higher washout percentage than others. Increasingin the washout percentage for the 12x-2x paste could be due to theslight low CaCO3 ratio than the 10x-2x paste. Summarizing thewashout test, supplementation of CaCO3 amount appropriately,i.e., respective Ca­Cit and CaCO3 amount of 10x and 2x; and 12xand 2x, improved the anti-washout property without impairing thepH of the PBS.Figure 4 shows the cell proliferation curve for the MG-63 cellscultured with the HAp/Col pastes. The 9x-1x paste was not usedfor the cell culture test because the pH of the medium turnedbasic after 72 h. Significant difference between the test groupsand TCPS group were found for each measurement. All fourcombinations showed good proliferation activity without anysignificant difference compared to the HAp/Col dense bodies,which shows very good biocompatibility in vivo.7)Figure 5 and 6 show the changes in the Ca2+ and PO43¹ ionconcentrations in the culture medium. At day 3, the culture mediaof test groups contained 3 times higher Ca2+ ion concentrationthan control TCPS due to the gradual release of Ca2+ ions fromCaCO3 and Ca­Cit. But, the ratio compared to the control de-creased as the culture period increased. In contrast, the Ca2+ ionFig. 4. In vitro analysis of the combination of additives on HAp/Colpaste. Data represent mean « SD for n = 5.Fig. 5. Calcium ion concentration of the culture medium after the cellculture test. Data represent mean « SD for n = 3.Manchinasetty et al.: Influences of combined supplementation of calcium citrate and calcium carbonate on injectable and anti-washouthydroxyapatite/collagen bone paste utilizing sodium alginateJCS-Japan582concentration decreased in HAp/Col dense body, due to theadsorption of Ca2+ ions on HAp/Col as reported by Sotomeet al.14) The concentration of PO43¹ ions continuously decreasedwith the culture period. The PO43¹ ion concentration in theculture media of test groups and HAp/Col dense body was lowcompared to control TCPS. Changes in Ca2+ and reduction inPO43¹ ion concentration in the culture medium could affecttheproliferation and differentiation of almost all cells; however, noquite suppression in the cell proliferation observed in the test andHAp/Col dense body group, which demonstrated that these ionconcentration changes had no critical effects on cell viability andproliferation. The cytocompatability of the pastes is as good asthe HAp/Col, clinically used material in Japan.These results suggest that the influence of the combinedsupplementation of Ca­Cit and CaCO3 on the injectable HAp/Col paste improved the anti-washout property and pH controlability without critical influences on biocompatibility.4. ConclusionsCombined supplementations of Ca­Cit and CaCO3 improvedthe anti-washout property and pH controllability of the injectableHAp/Col paste. The improvements were caused by a competitivereaction occurred coordinately in the pastes. From in vitro cellculture studies, all combinations showed good cytocompatibilitywithout any significant suppression of cell proliferations. Hence,the presently prepared HAp/Col pastes could be good candidatesfor injectable artificial bone, which has the potential for incor-poration into bone remodeling process.Acknowledgements: The first author would like to thank Mr.Sho Oshima (Ibaraki University, Japan) for his helpful support withthe characterization and SASTRA University, India for the financialsupport to carry out the semester abroad program at National Institutefor Materials Science, Japan.References1) C. J. Damien and J. R. Parsons, J. Appl. Biomater., 2, 187­208(1991).2) M. Komath, H. K. Varma and R. Sivakumar, Bull. Mater. Sci.,23, 135­140 (2000).3) M. Nilsson, L. Wielanek, J. S. Wang, K. E. Tanner and L.Lidgren, J. Mater. Sci-Mater. M., 14, 399­404 (2003).4) Y. Fukase, S. Wada, H. Uehara, M. Terakado, H. Sato and M.Nishiyama, J. Oral Sci., 40, 71­76 (1998).5) T. Konishi, M. Mizumoto, M. Honda, Y. Horiguchi, K. Oribe,H. Morisue, K. Ishii, Y. Toyama, M. Morio and M. Aizawa,J. Nanomater., 2013, 864374 (2013).6) T. Konishi, S. Takahashi, Z. Zhuang, K. Nagata, M. Mizumoto,M. Honda, Y. Takeuchi, H. Matsunari, H. Nagashima and M.Aizawa, J. Mater. Sci-Mater. M., 24, 1383­1394 (2013).7) M. Kikuchi, S. Itoh, S. Ichinose, K. Shinomiya and J. Tanaka,Biomaterials, 22, 1705­1711 (2001).8) M. Kikuchi, T. Ikoma, S. Itoh, H. N. Matsumoto, Y. Koyama,K. Tkakuda, K. Shinomiya and J. Tanaka, Compos. Sci.Technol., 64, 819­824 (2004).9) K. Shinomiya, M. Ishizuki, H. Morioka, S. Matasumoto, T.Nakamura, S. Abe and Y. Beppu, Seikei Geka-Shi, 63, 921­926(2012) [in Japanese].10) A. Kochi, M. Kikuchi, Y. Shirosaki, S. Hayakawa and A.Osaka, Key. Eng. Mat., 493–494, 689­692 (2012).11) K. Y. Lee and D. J. Mooney, Prog. Polym. Sci., 37, 106­126(2012).12) T. Sato, A. Kochi, Y. Shirosaki, S. Hayakawa, M. Aizawa, A.Osaka and M. Kikuchi, J. Ceram. Soc. Jpn., 121, 775­781(2013).13) T. Sato, M. Kikuchi and M. Aizawa, J. Mater. Sci-Mater. M.,28, 49 (2017).14) S. Sotome, T. Uemura, M. Kikuchi, S. Itoh, J. Tanaka, M.Takahashi, T. Tateishi and K. Shinomiya, Key. Eng. Mat., 218–220, 153­156 (2001).15) K. Ishikawa, Y. Miyamoto, M. Takechi, T. Toh, M. Kon, M.Nagayama and K. Asaoka, J. Biomed. Mater. Res., 36, 393­399 (1997).Fig. 6. Phosphate ion concentration of the culture medium after the cellculture test. Data represent mean « SD for n = 3.Journal of the Ceramic Society of Japan 125 [7] 579-583 2017 JCS-Japan583