Fabrication of Carbonate Apatite Block through a Dissolution ... - MDPI

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Fabrication of Carbonate Apatite Block through a Dissolution–Precipitation Reaction Using Calcium Hydrogen Phosphate Dihydrate Block as a Precursor Kanji Tsuru 1, *, Ayami Yoshimoto 1 , Masayuki Kanazawa 2 , Yuki Sugiura 1 , Yasuharu Nakashima 2 and Kunio Ishikawa 1 1

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Department of Biomaterials, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; [email protected] (A.Y.); [email protected] (Y.S.); [email protected] (K.I.) Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; [email protected] (M.K.); [email protected] (Y.N.) Correspondence: [email protected]; Tel.: +81-92-642-6345; Fax: +81-92-642-6348

Academic Editor: Enrico Bernardo Received: 27 January 2017; Accepted: 29 March 2017; Published: 31 March 2017

Abstract: Carbonate apatite (CO3 Ap) block, which is a bone replacement used to repair defects, was fabricated through a dissolution–precipitation reaction using a calcium hydrogen phosphate dihydrate (DCPD) block as a precursor. When the DCPD block was immersed in NaHCO3 or Na2 CO3 solution at 80 ◦ C, DCPD converted to CO3 Ap within 3 days. β-Tricalcium phosphate was formed as an intermediate phase, and it was completely converted to CO3 Ap within 2 weeks when the DCPD block was immersed in Na2 CO3 solution. Although the crystal structures of the DCPD and CO3 Ap blocks were different, the macroscopic structure was maintained during the compositional transformation through the dissolution–precipitation reaction. CO3 Ap block fabricated in NaHCO3 or Na2 CO3 solution contained 12.9 and 15.8 wt % carbonate, respectively. The diametral tensile strength of the CO3 Ap block was 2 MPa, and the porosity was approximately 57% regardless of the carbonate solution. DCPD is a useful precursor for the fabrication of CO3 Ap block. Keywords: bone replacement; carbonate apatite; calcium hydrogen phosphate dihydrate; dissolution–precipitation reaction

1. Introduction Bone apatite is different from hydroxyapatite (HAp; Ca10 (PO4 )6 (OH)2 ), but it is similar to carbonate apatite (CO3 Ap), which contains 6–9 wt % carbonate in the apatitic structure [1]. CO3 Ap is denoted as A-type or B-type on the basis of the substitution site of carbonate ions in the apatitic lattice. Type A CO3 Ap (CO3 -for-OH substitution) can be prepared at high temperature (1000 ◦ C) whereas B-type CO3 Ap (CO3 -for-PO4 substitution) can be prepared by precipitation or by hydrolysis at low temperature [1]. B-type CO3 Ap is a candidate for artificial bone substitute since its structure and crystallinity are similar to natural bone. However, B-type CO3 Ap decomposes thermally at sintering temperatures. As a result, sintering methods cannot be used for the fabrication of CO3 Ap bone substitute. In the era from the 1970s to 1980s, sintered HAp, which is free of carbonate, showed excellent tissue response and good osteoconductivity [2–4]. Therefore, sintered HAp has been widely used as an artificial bone substitute [5–7]. One major drawback of sintered HAp is its stability, and grafted HAp is hardly replaced by new bone. CO3 Ap block has been fabricated using dissolution–precipitation and precursors such as calcite [CaCO3 ] [8,9], gypsum [CaSO4 ·2H2 O] [10,11], and α-tricalcium phosphate (TCP; [α-Ca3 (PO4 )2 ]) [12–14]. Materials 2017, 10, 374; doi:10.3390/ma10040374

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When osteoclasts were incubated on the surface of CO3 Ap blocks, osteoclastic resorption pits similar to bone were observed [8]. Because of the absence and presence of osteoclastic resorption,2 ofsintered Materials 2017, 10, 374 10 HAp is not replaced by new bone, whereas CO3 Ap block is. CO3 Ap can upregulate differentiation of When osteoclasts incubated on the surface of CO 3Ap blocks, osteoclastic resorption pits similar than osteoblasts more thanwere HAp [15]. Moreover, CO3 Ap has demonstrated higher osteoconductivity to bone were observed [8]. Because of the absence and presence of osteoclastic resorption, sintered sintered HAp. HAp is not replaced by new bone, whereas CO3Ap block is. CO3Ap can upregulate differentiation of Although CO3 Ap block is a promising artificial bone replacement, the studies conducted so far to osteoblasts more than HAp [15]. Moreover, CO3Ap has demonstrated higher osteoconductivity than identify the ideal precursor have been limited. There are requirements for the precursor involved in the sintered HAp. fabrication of CO Ap block. First, a precursor should be a block. Compositional transformation from Although3 CO3Ap block is a promising artificial bone replacement, the studies conducted so far the precursor block occurs through a dissolution–precipitation reaction, maintaining 3 Ap to identifyto theCO ideal precursor have been limited. There are requirements for the precursor involved the macroscopic structure of the precursor. Second, the precursor should have at least one component of in the fabrication of CO3Ap block. First, a precursor should be a block. Compositional CO3 Ap such as calcium, or carbonate. the precursor be in a metastable phase transformation from phosphate the precursor to CO3Ap Third, block occurs throughshould a dissolution–precipitation reaction, maintaining the in macroscopic structure of the Second, the precursor should and have suitable solubility the solution used for theprecursor. dissolution–precipitation reaction. If have solubility leastitone component 3Ap such asCO calcium, phosphate or carbonate. Third, the precursor is tooatlow, takes too longoftoCO fabricate the Ap block from the precursor because of the rate of 3 should be in a metastable phase and have suitable solubility in the solution used for the dissolution– the dissolution–precipitation reaction. If the solubility is too high, the precursor cannot maintain its precipitation reaction. If solubility is too low, it takes too long to fabricate the CO3Ap block from the structure, and CO3 Ap powder is formed instead of CO3 Ap block. For example, calcium chloride precursor because of the rate of the dissolution–precipitation reaction. If the solubility is too high, cannot be a precursor because its solubility is too high even though it contains calcium. the precursor cannot maintain its structure, and CO3Ap powder is formed instead of CO3Ap block. Dicalcium dihydrate CaHPO O]solubility is a candidate for even the fabrication 4 ·2H2its For example,phosphate calcium chloride cannot[DCPD; be a precursor because is too high though it of CO3 Ap block. DCPD block can be fabricated from a setting reaction, i.e., DCPD-forming cement [16–19]. contains calcium. DCPD contains bothphosphate calcium and phosphate in its composition. blockfor may an unsuitable Dicalcium dihydrate [DCPD; CaHPO 4·2H2O] is DCPD a candidate thehave fabrication of solubility forblock. the compositional to aCO Ap through a dissolution–precipitation reaction CO3Ap DCPD block cantransformation be fabricated from setting reaction, i.e., DCPD-forming cement [16–19]. 3 DCPD bothof calcium phosphate in its composition. DCPD blocksuch may as have an unsuitable because thecontains solubility DCPDand is higher than that of other precursors calcite, α-TCP, and solubility for the compositional transformation to CO 3 Ap through a dissolution–precipitation gypsum. Besides, DCPD is an acidic calcium phosphate. For the compositional transformation to reaction because the solubility of DCPD is higher using than that of other block, precursors such as calcite, CO3 Ap through a dissolution–precipitation reaction a precursor the solution around the α-TCP, and gypsum. Besides, DCPD is an acidic calcium phosphate. For the compositional precursor block should be supersaturated with respect to CO3 Ap. An acidic environment is unsuitable transformation to CO3Ap through a dissolution–precipitation reaction using a precursor block, the for the liquid to be supersaturated with respect to CO3 Ap. solution around the precursor block should be supersaturated with respect to CO3Ap. An acidic In this study,isthe feasibility of the fabrication of CO3 Apwith block by compositional transformation environment unsuitable for the liquid to be supersaturated respect to CO3Ap. through dissolution–precipitation reaction using DCPD block as by a precursor wastransformation evaluated. DCPD In this study, the feasibility of the fabrication of CO3Ap block compositional blockthrough was prepared using a setting reaction of β-TCP and monocalcium phosphate monohydrate dissolution–precipitation reaction using DCPD block as a precursor was evaluated. DCPD [MCPM: blockCa(HPO was prepared using a setting reaction of β-TCP and monocalcium phosphate monohydrate 4 )2 ·H2 O]. [MCPM: Ca(HPO4)2·H2O].

2. Results 2. Results

Figure 1 summarizes the photographs of the set samples. Figure 1a shows the set sample resulting of MCPM the set samples. Figure 1a shows setsample sample resulting from the Figure setting1 summarizes reaction ofthe thephotographs β-TCP and mixture. Figure 1b isthethe obtained by from the setting reaction of the β-TCP and MCPM◦ mixture. Figure 1b is the sample obtained by immersion of the set sample in 2 M NaHCO3 at 80 C for 14 days. Figure 1c is the sample obtained immersion of the set sample in 2 M NaHCO3 at 80 °C for 14 days. Figure 1c is the sample obtained by by immersion of the set sample in 2 M Na2 CO3 at 80 ◦ C for 14 days. Samples maintained their immersion of the set sample in 2 M Na2CO3 at 80 °C for 14 days. Samples maintained their macroscopic macroscopic structure after the immersion in carbonate solutions regardless of the carbonate solution. structure after the immersion in carbonate solutions regardless of the carbonate solution.

Figure 1. Photographs of set samples. (a) Set sample made with the setting reaction of the

Figure 1. Photographs of set samples. (a) Set sample made with the setting reaction of the β-tricalcium β-tricalcium phosphate and monocalcium phosphate monohydrate mixture, and samples obtained phosphate and monocalcium phosphate monohydrate mixture, and samples obtained by immersion in by immersion in (b) 2 M NaHCO3 and (c) 2 M Na2CO3 solutions at 80 °C for 14 days. (b) 2 M NaHCO3 and (c) 2 M Na2 CO3 solutions at 80 ◦ C for 14 days.

Figure 2 summarizes representative scanning electron microscope (SEM) and transmission electron microscope (TEM) images of a set sample made with the setting reaction of the β-TCP and

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Figure 2 summarizes representative scanning electron microscope (SEM) and transmission electron microscope β-TCP Materials 2017, 10, 374 (TEM) images of a set sample made with the setting reaction of the 3 of 10 Materials 2017, 10, 374 3 of 10 ◦ and MCPM mixture, after the sample was immersed in 2 M NaHCO3 at 80 C for 14 days, and MCPM mixture, after thedays. sample was immersed immersed in 22 M M maintained NaHCO33 at at 80 80 °C for for 14 14 days, days, and and M as 2 M Na at 80 ◦ Cafter for the 14 Although the samples their macroscopic structure 2 CO3mixture, MCPM sample was in NaHCO °C 22 M Nain 2CO3 at 80 °C for 14 days. Although the samples maintained their macroscopic structure as shown shown Figure 1, for the14 morphology of the crystalsmaintained was different and after immersion Na 2CO 3 at 80 °C days. Although the samples their before macroscopic structure as shownin the in Figure 1, the morphology of the crystals was different before and after immersion in the carbonate in Figure 1, the morphology of the crystals and of after in the carbonate carbonate solution. The sample made withwas thedifferent settingbefore reaction theimmersion β-TCP and MCPM mixture solution. The The sample made made with with the the setting setting reaction reaction of of the the β-TCP β-TCP and and MCPM MCPM mixture showed showed solution. showed plate-likesample crystals, which are similar to the typical morphology of DCPDmixture crystals (Figure 2a). plate-like crystals, crystals, which which are are similar similar to to the the typical typical morphology morphology of DCPD DCPD crystals crystals (Figure (Figure 2a). 2a). When When plate-like When thesample sample was immersed inM2 M NaHCO at 80 ◦ C forof14 days, crystals with two different the was immersed in 2 NaHCO 3 at 380 °C for 14 days, crystals with two different the sample was immersed in 2 M NaHCO3 at 80 °C for 14 days, crystals with two different morphologies were present in the sample (Figure 2b-SEM). One crystal showed a plate-like structure morphologies were were present present in in the the sample sample (Figure (Figure 2b-SEM). 2b-SEM). One One crystal crystal showed showed aa plate-like plate-like structure structure morphologies similar to DCPD crystals. On the surface of the plate-like crystals, small needle-like crystals similar to DCPD crystals. On the surface of the plate-like crystals, small needle-like crystals were were similar to DCPD crystals. On the surface of the plate-like◦crystals, small needle-like crystals were present. However, when immersed at80 80°C for14 days, polygon-like crystals present. However, when immersedinin in222M MNa Na222CO at 80 °CCfor for 1414 days, polygon-like crystals were were present. However, when immersed M Na CO333at days, polygon-like crystals were formed (Figure 2c-SEM). The TEM micrographs supported the results of the SEM observation. formed (Figure 2c-SEM). The TEM micrographs supported the results of the SEM observation. formed (Figure 2c-SEM). The TEM micrographs supported the results of the SEM observation.

Figure 2. Representative SEM and TEM images of samples. (a) Set sample made with the setting Figure 2. Representative SEM and TEM images of samples. (a) Set sample made with the setting

Figure 2. Representative SEMphosphate and TEMand images of samples. (a) Set sample made withand theset setting reaction of the β-tricalcium monocalcium phosphate monohydrate mixture reaction of the β-tricalcium phosphate and monocalcium phosphate monohydrate mixture and set samples immersed in (b) 2phosphate M NaHCO3and and (c) 2 M Na2CO3 solutions at 80 °C for 14 days.mixture and set reaction of the β-tricalcium monocalcium phosphate monohydrate samples immersed in (b) 2 M NaHCO3 and (c) 2 M Na2CO3 solutions at 80 °C for 14 days. samples immersed in (b) 2 M NaHCO3 and (c) 2 M Na2 CO3 solutions at 80 ◦ C for 14 days. Figure 33 summarizes summarizes the the powder powder XRD XRD patterns. patterns. In In addition addition to to the the XRD XRD patterns patterns of of β-TCP β-TCP Figure powder, MCPM powder, β-TCP-MCPM mixture powder, and the set sample made from the mixture powder, powder, powder, and thetoset sample from mixture Figure 3MCPM summarizes theβ-TCP-MCPM powder XRDmixture patterns. In addition the XRD made patterns ofthe β-TCP powder, of β-TCP β-TCP and and MCPM, MCPM, aa standard standard DCPD DCPD pattern pattern is is shown shown to to facilitate facilitate comparison. comparison. The The mixture mixture of of of MCPM powder, β-TCP-MCPM mixture powder, and the set sample made from the mixture of β-TCP β-TCP and MCPM became DCPD when exposed to water during the setting reaction. β-TCP and MCPM became DCPD when exposed to water during the setting reaction. and MCPM, a standard DCPD pattern is shown to facilitate comparison. Theismixture Hereafter, the set set sample made with with the mixture mixture of β-TCP β-TCP and MCPM MCPM referred of to β-TCP as the the and Hereafter, the sample made the of and is referred to as MCPM became DCPD when exposed to water during the setting reaction. DCPD block. block. DCPD

Figure 3. X-ray diffraction patterns of (a) β-tricalcium phosphate (TCP) powder; (b) monocalcium

Figure 3. X-ray diffraction patternsofof(a) (a)β-tricalcium β-tricalcium phosphate (TCP) powder; (b) monocalcium Figure 3. X-ray diffraction patterns phosphate (TCP) powder; (b) monocalcium phosphate monohydrate (MCPM) powder; (c) β-TCP and MCPM mixed powder; (d) set sample phosphate monohydrate (MCPM) powder; β-TCP and MCPM mixedpowder; powder;(d) (d)set setsample samplemade phosphate monohydrate (MCPM) powder; (c) (c) β-TCP and MCPM mixed made from the mixture of β-TCP and MCPM; and (e) calcium hydrogen phosphate dihydrate made from the mixture of β-TCP and MCPM; and (e) calcium hydrogen phosphate dihydrate from powder the mixture of β-TCP and MCPM; and (e) calcium hydrogen phosphate dihydrate powder was was used as a reference. powder was used as a reference. used as a reference.

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Hereafter, the set sample made with the mixture of β-TCP and MCPM is referred to as the DCPD block. Figure 4 summarizes the XRD patterns of the DCPD block before and after immersion in 2 M Figure 4 summarizes the XRD patterns of the DCPD block before and after immersion in 2 M NaHCO3 or 2 M Na2CO3 at 80 °C for up to 14 days. After 3 days of immersion, peaks similar to NaHCO3 or 2 M Na2 CO3 at 80 ◦ C for up to 14 days. After 3 days of immersion, peaks similar to CO3Ap were detected, and the peaks assigned to DCPD disappeared. Furthermore, the peaks CO3 Ap were detected, and the peaks assigned to DCPD disappeared. Furthermore, the peaks assigned assigned to β-TCP (indicated by the open circles in Figure 4) were also detected regardless of sodium to β-TCP (indicated by the open circles in Figure 4) were also detected regardless of sodium carbonate carbonate solution. The peak height of β-TCP corresponding to the amount of β-TCP decreased with solution. The peak height of β-TCP corresponding to the amount of β-TCP decreased with immersion immersion time. In the case of immersion in the NaHCO3 solution, the β-TCP peaks remained even time. In the case of immersion in the NaHCO3 solution, the β-TCP peaks remained even after 14 after 14 days. However, in the case of immersion in the Na2CO3 solution, the peak height of β-TCP, days. However, in the case of immersion in the Na2 CO3 solution, the peak height of β-TCP, which which was lower than that for NaHCO3 immersion, decreased with time and disappeared within was lower than that for NaHCO3 immersion, decreased with time and disappeared within 14 days. 14 days. Therefore, conversion to CO3Ap was faster in the case of Na2CO3 immersion than in the case Therefore, conversion to CO3 Ap was faster in the case of Na2 CO3 immersion than in the case of of NaHCO3 immersion. In addition, the CO3Ap peaks became broad so that a low-crystalline CO3Ap NaHCO In addition, the CO3 Ap peaks became broad so that a low-crystalline CO3 Ap 3 immersion. was obtained in this method. was obtained in this method.

Figure 4. X-ray diffraction patterns of calcium hydrogen phosphate dihydrate block (a) before and Figure 4. X-ray diffraction patterns of calcium hydrogen phosphate dihydrate block (a) before and for up to 14 days. after immersion in (b–d) 2 M NaHCO3 and (e–g) 2 M Na2CO3 solutions at 80 °C after immersion in (b–d) 2 M NaHCO3 and (e–g) 2 M Na2 CO3 solutions at 80 ◦ C for up to 14 days. XRD patterns of (h) β-tricalcium phosphate and (i) CO3Ap are listed as references. XRD patterns of (h) β-tricalcium phosphate and (i) CO3 Ap are listed as references.

Figure 5 summarizes the FTIR spectra of the DCPD block (a) before and after immersion in 5 summarizes the FTIR spectra of the DCPD block (a) before and after immersion in (b) (b) 2 Figure M NaHCO 3 at 80 °C for up to 14 days, and after immersion in (c) 2 M Na2CO3 at 80 °C for up to ◦ C for up to 14 days, and after immersion in (c) 2 M Na CO at 80 ◦ C for up to 214Mdays. NaHCO at 80 2 3 The3 FTIR spectrum of (d) CO3Ap is shown for comparison. The frequencies at 567, 606, 14 days. The FTIR spectrum of (d) CO Ap is shown for comparison. The frequencies at 567, 1042, 3 3− −1 1042, and 1092 cm are assigned to PO4 groups [20], the frequencies at 875, 1418, and 1474606, cm−1 are −1 are assigned to PO 3− groups [20], the frequencies at 875, 1418, and 1474 cm−1 are and 1092 cm 4 assigned to CO32− groups [21], and the frequencies at 640 cm−1 are assigned to OH− groups [20]. FTIR − groups [21], and the frequencies at 640 cm−1 are assigned to OH− groups [20]. assigned to CO3 2after spectra obtained immersed in sodium carbonate solutions (Figure 5b,c) were similar to those of FTIR spectra obtained after immersed in those sodium solutions 5b,c) weredue similar to CO3Ap (Figure 5d) and different from ofcarbonate DCPD (Figure 5a).(Figure A larger band to low those of CO Ap (Figure 5d) and different from those of DCPD (Figure 5a). A larger band due to crystallinity 3was observed when the DCPD block was immersed in the carbonate solutions. The low crystallinity was observed when the DCPD block was immersed in the carbonate solutions. The band at 1413 cm−1 observed in the obtained CO3Ap is reported to be assigned to B-type CO3Ap [1]. band at 1413 cm−1 observed in −the obtained reported be obtained assigned CO to B-type CO3 Ap [1]. 3 Ap isAs No peaks corresponding to OH groups wereCO present. a result,tothe 3Ap is expected to − groups were present. As a result, the obtained CO Ap is expected to No peaks corresponding to OH 3 be AB-type CO3Ap. be AB-type CO3 Ap.

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Figure 5. Fourier Fouriertransform transform infrared spectra of calcium hydrogen phosphate dehydrate block Figure 5. infrared spectra of calcium hydrogen phosphate dehydrate block (a) before 3 and (c) 2 M Na2CO3 solutions at 80◦°C for 14 days; (a) before and after immersion in (b) 2 M NaHCO and after immersion in (b) 2 M NaHCO3 and (c) 2 M Na2 CO3 solutions at 80 C for 14 days; (d) (d) Spectrum Spectrum of of CO CO3Ap Apisisused usedas asaareference. reference. 3

Figure 5. Fourier transform infrared spectra of calcium hydrogen phosphate dehydrate block 3 and (c)blocks 2 M Na2after CO3 solutions at 80 °C 14 NaHCO days; before and afterthe immersion in (b)content 2 M NaHCO Table(a) 1 summarizes carbonate of DCPD immersion in for 2M 3 and Table(d) 1 Spectrum summarizes the carbonate content of DCPD blocks after immersion in 2 M NaHCO3 and of CO 3Ap is used as a reference. 2 M Na2CO3 at 80 °C for up to 14 days. Both samples contained CO3. The carbonate contents of the 2 M Na CO at 80 ◦ C for up to 14 days. Both samples contained CO . The carbonate contents of the 2

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DCPD block immersed in 2 M NaHCO3 and Na2CO3 at 80 °C for 14 days were 12.9% ± 0.5% and DCPD block immersed in 2the M carbonate NaHCO3content and Na CO3 atblocks 80 ◦ Cafter forimmersion 14 days were 12.9% ± 30.5% Table 1 summarizes of2DCPD in 2 M NaHCO and and 15.8% ± 0.9%, respectively. M0.9%, Na2CO 3 at 80 °C for up to 14 days. Both samples contained CO3. The carbonate contents of the 15.8%2 ± respectively. DCPD block immersed in 2 M NaHCO3 and Na2CO3 at 80 °C for 14 days were 12.9% ± 0.5% and Table Carbonate content of DCPD blocks after immersion in 2 M NaHCO3 and Na2CO3 at 80 °C for 15.8%1. 0.9%, respectively. Table 1.± Carbonate content of DCPD blocks after immersion in 2 M NaHCO3 and Na2 CO3 at 80 ◦ C for 14 days. 14 days. Table 1. Carbonate content of DCPD blocks after immersion in 2 M NaHCO3 and Na2CO3 at 80 °C for Solution CO2 Contents (wt %) 14 days.

Solution NaHCO3 Solution NaHCO Na 2CO 3 3 NaHCO 3 Na2 CO3 Na2CO3

2 Contents (wt %) 12.9CO ± 0.5 CO2 Contents %)± 0.5 12.9 15.8 ± 0.9(wt 12.9 ± 0.5 15.8 ± 0.9 15.8 ± 0.9

Figure 6 summarizes the DTS values of the DCPD block before and after immersion in the carbonate solutions. No statically significant difference was observed before and after immersion Figure 6 summarizes thethe DTS block beforeand and after immersion in the Figure 6 summarizes DTSvalues values of of the the DCPD DCPD block before after immersion in the regardless of the carbonate solution. carbonate solutions. NoNo statically wasobserved observed before after immersion carbonate solutions. staticallysignificant significant difference difference was before andand after immersion regardless of the solution. regardless of carbonate the carbonate solution.

Figure 6. Diametral tensile strengthsthe thecalcium calcium hydrogen hydrogen phosphate dihydrate block (a) before and and Figure 6. Diametral Diametral tensile strengths phosphate dihydrate block (a)before before Figure 6. tensile strengths the calcium hydrogen phosphate dihydrate block (a) and after immersion in (b) 2 M NaHCO3 and (c) 2 M Na2CO3 solutions at 80 °C ◦for 14 days. after immersion immersion in in (b) (b) 22 M M NaHCO NaHCO33and and(c) (c)22M MNa Na22CO CO3 3solutions solutionsatat8080°CCfor for1414days. days. after

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Figure 77summarizes summarizes porosity of DCPD the DCPD block before and after immersion in the Figure thethe porosity of the block before and after immersion in the carbonate carbonate solutions. Theofporosity of the DCPD 37.4% ± 3.3%. After immersion in the solutions. The porosity the DCPD block was block 37.4% was ± 3.3%. After immersion in the carbonate carbonateporosity solution, porositytoincreased 56.5% ± 1.9%3 solution) (NaHCO3and solution) and 56.6%± 1.8% (Na2CO3 solution, increased 56.5% ± to 1.9% (NaHCO 56.6%± 1.8% (Na2 CO 3 solution). solution). Nosignificant statically significant wasbetween observedthe between the solutions. carbonate solutions. No statically difference difference was observed carbonate

Figure 7. Porosity of the calcium hydrogen phosphate dihydrate block (a) before and after immersion ◦ Cfor in (b) 2 M NaHCO33 and (c) 2 M Na22CO CO33solutions solutionsat at80 80°C for14 14days. days.

3. Discussion 3. Discussion The results results obtained obtained in in this this study study demonstrate demonstrate that that CO CO33Ap fabricated though though aa The Ap block block can can be be fabricated dissolution–precipitation reaction using DCPD block as a precursor. The DCPD block satisfied the dissolution–precipitation reaction using DCPD block as a precursor. The DCPD block satisfied the requirements for for fabricating fabricating the the CO CO33Ap Ap block. block. requirements An ideal precursor is a block because the macroscopic macroscopic structure structure is is maintained maintained during during the the An ideal precursor is a block because the dissolution–precipitation reaction. As shown in Figure 1a, the DCPD block can be made by a setting dissolution–precipitation reaction. As shown in Figure 1a, the DCPD block can be made by a reactionreaction of a mixture of β-TCPofand MCPM a setting DCPD-forming cement. The SEM setting of a mixture β-TCP andor MCPM or areaction setting of reaction of DCPD-forming cement. observation shown in Figure 2a reveals that precipitated DCPD crystals interlock with each other The SEM observation shown in Figure 2a reveals that precipitated DCPD crystals interlock with during setting. Thesetting. settingThe reaction the DCPD block is also a dissolution–precipitation reaction. each other during settingofreaction of the DCPD block is also a dissolution–precipitation This microporous structure may be an ideal precursor for CO 3Ap fabrication through a dissolution– reaction. This microporous structure may be an ideal precursor for CO3 Ap fabrication through a precipitation reaction because the because dissolution of the microporous block is faster than that ofthan a dense dissolution–precipitation reaction the dissolution of the microporous block is faster that block, and block, there is space foristhe formation of CO3Ap crystals. of a dense and there space for the formation of CO3 Ap crystals. The setting setting time time of The of the the DCPD-forming DCPD-forming cement cement was was very very short short in in the the absence absence of of aa retarder. retarder. Although the setting time can be regulated by adding retarders such as citric acid, pyrophosphate, Although the setting time can be regulated by adding retarders such as citric acid, pyrophosphate, or sulfuric sulfuricacid, acid,no noretarder retarder was introduced to the mixture of β-TCP and MCPM the present or was introduced to the mixture of β-TCP and MCPM in thein present study study to simplify the precursor. to simplify the precursor. DCPD contains containsboth both calcium phosphate; thus,carbonate only carbonate is for required for the DCPD calcium andand phosphate; thus, only is required the fabrication fabrication of the CO 3Ap block. Therefore, the DCPD block was immersed in NaHCO3 or Na2CO3 of the CO3 Ap block. Therefore, the DCPD block was immersed in NaHCO3 or Na2 CO3 solution in solution in study. the present study. The solution has a closewith relationship with Although, the solubility. the present The solution condition hascondition a close relationship the solubility. the Although, the solubilities DCPD and in the NaHCO 3 and Na2CO3 solutions are higher than other solubilities of DCPD in the of NaHCO Na CO solutions are higher than other precursors such as 3 2 3 precursors such as calcite, calcium sulfate dihydrate, or α-TCP, the DCPD block was suitable for calcite, calcium sulfate dihydrate, or α-TCP, the DCPD block was suitable for CO3 Ap block fabrication CO3Ap block because it was not dissolved during the reaction, and the because it wasfabrication not dissolved during the reaction, and the macroscopic structure wasmacroscopic retained as structure was retained as shown in Figure 1b,c. Because of the high solubility, the DCPD phase shown in Figure 1b,c. Because of the high solubility, the DCPD phase disappears as early as 1 day of disappears as early as 1 day of immersion regardless of the carbonate solution. Faster compositional immersion regardless of the carbonate solution. Faster compositional transformation from DCPD to transformation from DCPD to CO3Ap is reasonable because this reaction proceeds through a CO 3 Ap is reasonable because this reaction proceeds through a dissolution–precipitation mechanism. dissolution–precipitation mechanism. β-TCP wascarbonate present 1solutions day after in thephase. carbonate β-TCP was present 1 day after immersion in the asimmersion an intermediate The solutions as an intermediate phase. The amount of β-TCP was higher when the DCPD block amount of β-TCP was higher when the DCPD block was immersed in NaHCO3 solution thanwas in immersed in NaHCO 3 solution than in Na2CO3 solution. The amount of β-TCP decreased with Na2 CO3 solution. The amount of β-TCP decreased with immersion time and disappeared completely immersion time and disappeared completely when the DCPD block was immersed in Na2CO3 solution for 2 weeks. However, β-TCP remained, even after 2 weeks when immersed in NaHCO3 solution. This difference may be because of the pH of the solution. In other words, β-TCP is more

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when the DCPD block was immersed in Na2 CO3 solution for 2 weeks. However, β-TCP remained, even after 2 weeks when immersed in NaHCO3 solution. This difference may be because of the pH of the solution. In other words, β-TCP is more stable at neutral pH. The appearance of β-TCP as an intermediate phase demonstrates the possibility to use β-TCP block as a precursor for CO3 Ap block fabrication even though its solubility is much lower than that of α-TCP. Moreover, the β-TCP block may be fabricated in the solution. The β-TCP block fabricated in the solution may have a higher osteoconductivity than β-TCP block fabricated using a sintering process. Different pH values of the carbonate solution cause different crystal morphologies as shown in Figure 2b,c. Slower dissolution of DCPD in NaHCO3 solution than in Na2 CO3 solution may be the reason for this difference. In other words, DCPD crystals, shown in Figure 2a, dissolve quickly, and the solution around DCPD is highly supersaturated with respect to CO3 Ap, leading to the formation of more CO3 Ap nuclei, increasing CO3 Ap formation. When the DCPD block is immersed in NaHCO3 solution, DCPD dissolves slowly and results in a less supersaturated solution with respect to CO3 Ap. Therefore, the precipitation of CO3 Ap occurs only on the surface of DCPD crystals, and CO3 Ap crystals maintain the crystal structure of DCPD. Since the CO3 Ap block was prepared through a dissolution-precipitation reaction at low temperature (80 ◦ C), the FTIR spectra of the obtained CO3 Ap block had a broad band, indicating low crystalline CO3 Ap that was also confirmed by XRD results. The large band made it difficult to quantitatively determine the types of CO3 Ap. The band at 1413 cm−1 observed in the obtained CO3 Ap is reported to be assigned to B-type CO3 Ap [1]. Therefore it would be B-type CO3 Ap. However, no peaks corresponding to OH− groups were present in the spectra of the obtained CO3 Ap. As a result, not only B-type but also A-type might co-exist in the obtained CO3 Ap. Moreover, a broad band near 875 cm−1 assigned to the CO3 2− group consists of three components, such as A-type (878 cm− 1 ), B-type (871 cm− 1 ) and CO3 2 − formed from apatitic lattice (866 cm− 1 ) [22]. Based on the results, wealso have to consider the formation of CO3 2 − from apatitic lattice such as that adsorbed on the surface of CO3 Ap crystals. The mechanical strength of the CO3 Ap block in terms of DTS was higher (p < 0.05) when the DCPD block was immersed in NaHCO3 solution due to the crystal structure of CO3 Ap. Plate-like CO3 Ap shows high interlocking among the crystals. Although there was a slight difference in mechanical strength, there were no differences in porosity. The porosity of the CO3 Ap block was approximately 57% regardless of the carbonate solution, which was lower than that of the DCPD block. The Ca/P ratio of DCPD is lower than that of CO3 Ap, which has a Ca/P ratio around 2. Thus, DCPD needs to release PO4 and gain CO3 to transform its composition to CO3 Ap. A high porosity indicates that the amount of released PO4 is higher than the amount of incorporated CO3 from the solution. Therefore, CO3 Ap fabricated by compositional transformation through a dissolution–precipitation reaction using a precursor maintains its macroscopic structure but cannot maintain its microscopic structure. 4. Materials and Methods 4.1. Preparation of the DCPD Block The DCPD block was formed by the setting reaction of β-TCP and MCPM. β-TCP powder (Taihei, Osaka, Japan) and MCPM powder (Sigma–Aldrich Co., Saint Louis, MO, USA) were mixed with methanol (Wako Pure Chemical, Osaka, Japan) at a Ca/P molar ratio of 1.0. The methanol was allowed to evaporate at room temperature, and the mixture was placed into a split plastic steel mold 6 mm in diameter and 3 mm in height. Water was added dropwise until a water to powder weight ratio of 0.001 was reached. The samples were kept at 100% humidity for 24 h prior to testing. 4.2. Compositional Transformation from the Precursor Block to the CO3 Ap Block The obtained DCPD block was immersed in 2 M NaHCO3 or 2 M Na2 CO3 solution at 80 ◦ C for up to 14 days. In this treatment, ten DCPD blocks were immersed in each sodium carbonate solution

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of 500 mL. After the treatment, the DCPD blocks were removed from the sodium carbonate solution and rinsed with distilled water. 4.3. Powder X-ray Diffractometry The crystal phase of the obtained samples was detected by powder X-ray diffraction (XRD) analysis. The specimen was ground into a fine powder and used for the analysis. XRD patterns were recorded with an X-ray diffractometer (D8 Advance, Bruker AXS GmbH., Karlsruhe, Germany) using monochromatized X-ray (CuKα: λ = 0.1542 nm) operating at the condition of 40 kV and 40 mA. The diffraction angle was continuously scanned from 10◦ to 60◦ in 2θ at a scanning rate of 2◦ /min. A range of 10◦ –40◦ is shown in the figures because no relevant peaks were observed in the excluded region. 4.4. Fourier Transform Infrared Spectroscopy Fourier transform infrared (FTIR) spectroscopy was performed with an FTIR spectrometer (FT/IR-6200, JASCO, Tokyo, Japan) using the KBr method over a wavenumber range of 400–2000 cm−1 . A spectral resolution of 4 cm−1 was employed to examine structural changes. 4.5. Electron Microscopy The surface morphology of the obtained samples was observed by a scanning electron microscope (SEM; S-3400N, Hitachi High-Technologies Co., Tokyo, Japan) at 15 kV of accelerating voltage after gold–palladium coating by a magnetron sputtering machine (MSP-1S, Vacuum Device Co., Ibaraki, Japan). The fine structure of the obtained samples was observed by a transmission electron microscope (TEM; JEM-1400Plus, JEOL Co., Tokyo, Japan) at 100 kV of accelerating voltage. 4.6. Porosity Measurement The porosity the obtained specimen was calculated using the bulk density of the sample (dsample ) and the theoretical density of HAp (dHAp 3.16 g/cm3 ) [23], as shown in Equation (1). Porosity (%) = dHAp − dsample /dHAp × 100

(1)

4.7. Carbonate Contents Carbonate contents were estimated from the wt % of carbon in the CO3 Ap block. A CHN coder (MT-6; Yanako Analytical Instruments, Kyoto, Japan) was used to analyze the wt % of carbon. 4.8. Mechanical Strength Measurement The mechanical strength of disk-shaped samples was evaluated in terms of their diametral tensile strength (DTS). After drying the samples at 60 ◦ C for 24 h, their diameter and thickness were measured using a micrometer (MDC-25MU, Mitutoyo Co. Ltd., Kawasaki, Japan), and the samples were weighed using a microbalance. The samples were crushed with a universal testing machine (AGS-J, Shimadzu, Kyoto, Japan) at a constant crosshead speed of 10 mm/min. The mean DTS value for eight samples was calculated and expressed as mean ± standard deviation. 4.9. Statistical Analysis For statistical analysis, one-way analysis of variance and Fisher’s LSD method, as a post-hoc test, were performed using Kaleida Graph 4. We consider that p < 0.05 is statistically significant. 5. Conclusions CO3 Ap block was fabricated using DCPD block as a precursor by immersing the block in carbonate solutions. β-TCP forms as an intermediate phase during the transformation of the DCPD block to the

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CO3 Ap block by compositional transformation through a dissolution–precipitation reaction. Further studies are awaited based on the results obtained in this initial study. Acknowledgments: This study was supported, in part, by the Strategic Promotion of Innovative Research and Development Program (grant number 16im0502004h) from the Japan Agency for Medical Research and Development, and the Grant-in-Aid for Scientific Research (B) (grant number 15H05035) from the Japanese Society for Promotion of Science and Research. Author Contributions: K.T. and K.I. conceived and designed the experiments; M.K. and A.Y. performed the experiments and analyzed the data; Y.S. contributed TEM observations; K.T. and K.I. wrote the manuscript; and K.T., Y.N. and K.I. discussed the experiments and the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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