- Email: [email protected]
β-tricalcium phosphate bioceramics
Materials Science and Engineering C 59 (2016) 1007–1015
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Preparation, characterization and in vitro dissolution behavior of porous biphasic α/β-tricalcium phosphate bioceramics Lu Xie a, Haiyang Yu a,⁎, Yi Deng b,c,⁎⁎, Weizhong Yang b, Li Liao b, Qin Long b a b c
State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University, Chengdu 610065, China School of Chemical Engineering, Sichuan University, Chengdu 610065, China Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
a r t i c l e
i n f o
Article history: Received 13 August 2015 Received in revised form 28 October 2015 Accepted 13 November 2015 Available online 14 November 2015 Keywords: Porous α/β-tricalcium phosphate Bioceramic Dissolution Bone tissue engineering
a b s t r a c t The ideal bone tissue engineering scaffolds are long-cherished with the properties of interconnected macroporous structures, adjustable degradation and excellent biocompatibility. Here, a series of porous α/βtricalcium phosphate (α/β-TCP) biphasic bioceramics with different phase ratios of α-TCP and β-TCP were successfully synthesized by heating an amorphous calcium phosphate precursor. The chemical and morphological characterization showed that α- and β-TCP phases co-existed in the α/β-TCP bioceramics and they had interconnected pore structures with size between 200 and 500 μm. The in vitro dissolution behavior and bioactivity of the dual α/β-TCP were also probed in static and dynamic SBF for the first time. The results revealed that α/β-TCP scaffolds had good in vitro bioactivity, as the formation of bone-like apatite layers was induced on the scaffolds after mineralization in SBF. Moreover, dissolution rate of α/β-TCP bioceramics in dynamic environment was higher than that under static condition. Compared with monophasic TCP ceramics, these porous α/β-TCP bioceramics displayed a tailored dissolution rate proportionate to the TCP content (α and β) in the materials. Further, the degradation profile of porous α/β-TCP was well-described by Avrami equation. The porous dual α/β-TCP bioceramics with controllable degradation behavior hold great potential to be applied in bone tissue engineering as bone substitutes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The incidence of bone deficiencies associated with osteoporosis, trauma, congenital malformations, and surgical resections is rising substantially so that governments have to continuously escalate budget to balance the growing healthcare expenditures every year [1,2]. Current treatments of large bone defects, which rely on the use of autologous grafts (the “gold standard” of bone substitution) and allograft transplantation [3], have limited clinical potential due to their inherent limitations, such as donor site morbidity, available quantities, and host immune rejection. These imperfections have already led to synthetic bone grafts becoming a powerful alternative in replacing autologous grafts. On the other hand, one of the actual and much-needed demands in orthopedics is the clinical availability of degradable implants [4–6]. In some clinical applications, such as fracture treatment, permanent metal or polymer implants are not necessary or even disadvantageous, and a
⁎ Correspondence to: H. Yu, State Key Laboratory of Oral Diseases, Sichuan University, No. 24 South Section 1, Yihuan Road, Chengdu 610065, China. ⁎⁎ Correspondence to: Yi Deng, Academy for Advanced Interdisciplinary Studies, Peking University, No. 5 Yiheyuan Road, Haidian District, Beijing, 100871, China. E-mail addresses: [email protected] (H. Yu), [email protected] (Y. Deng).
http://dx.doi.org/10.1016/j.msec.2015.11.040 0928-4931/© 2015 Elsevier B.V. All rights reserved.
temporary implant would be much more suitable. Temporary implants made of biodegradable materials are destined to dissolve and degrade postoperatively synchronized with bone healing periods, so that a second surgery for implant removal is escapable, obviating surgery and associated cost, as well as unnecessary health risks and pain for patients. Thus, the design and construction of controllable degradable materials in the body environment is still an imperative and hot research area in the application of bone tissue engineering. Tricalcium phosphate (Ca3(PO4)2, TCP), one member of the calcium phosphate ceramics family, has been considered as an ideal temporary scaffold used for bone/dental repair replacing autogenous bone, because of its close chemical resemblance to biological apatite present in human hard tissues [7–9]. TCP has four polymorphic forms [10]: α, β, α′ and γ. β-tricalcium phosphate (β-TCP) is stable at room temperature and reconstructively transforms into α-tricalcium phosphate (α-TCP) between 1185 and 1430 °C, which is metastably retained during the cooling until room temperature; α′- and γ-TCP form above 1430 °C and under high pressures, respectively, but they lack interest, since it is difficult for them to attain a single phase owing to their metastability [11]. Nevertheless, α-TCP has restricted biomedical applications and is mainly employed in bone cement because of its high resorbability and reactivity [12,13]. It is reported that the solubility and/or degradation rate of α-TCP are much higher than those of β-TCP [14]. In body fluids,
1008
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015
the higher local Ca2+ and PO3− concentration caused by the rapid dis4 solution of α-TCP bioceramic might hamper the bone formation [15]. In contrast, β-TCP is famous for its remarkable osteoconductivity and biocompatibility. Lu et al. previously studied the degradation mechanism of β-TCP in bone and found a new lamellar bone formation following direct contact with the β-TCP implant and surrounding osteoblasts after a few weeks of implantation [16]. It was progressively absorbed in vivo by a cell-mediated process and replaced by new bone, reported by Kondo et al. [17] and Ogose et al. [18]. However, β-TCP also presents poor reactivity as a pure material, because it suffers from drawbacks such as slow in vivo bone formation and less-than-ideal degradation/ solubility. Furthermore, the difficulty in matching the degradation and the growth rates of new bone is a tough challenge for the clinical application of TCP bioceramics, because the degradation rate of a single calcium phosphate phase cannot be modulated. The degradation rate would be broadened if the two polymorphs of the TCP ceramics mentioned above are combined. Complete biodegradation within 4–6 months is required for biomaterials [19] in clinical trials of bone regeneration and repair. The degradation rate of the materials should be regulated to conform to the clinical demands, therefore, the investigation of degradability and understanding of the dissolution kinetic of TCP biomaterials remain desired. Generally, in vitro degradation performance of materials is often studied in physiological saline [20], phosphate buffered saline (PBS) solution [21,22], Hanks' balanced salt solution [23], and simulated body fluid (SBF) [24,25]. Among these solutions, the chemical composition and ion concentration of SBF are nearly equal to that of human blood plasma, so SBF is extensively applied in assessment for the dissolution behavior and in vitro bioactivity of a biomaterial [26]. Currently, although there is great progress in the preparation of a variety of βTCP-based biocomposites including PLGA/β-TCP [27], gelatin/β-TCP [28], PLLA/β-TCP composite [29], and HA/β-TCP biphasic calcium phosphate (called BCP) [30] with the goal of modulating the degradation rate, the degradation rate range of these β-TCP-based composites still cannot satisfy the clinical standards, and it is a challenge to synthesize highly degradable calcium phosphates. New degradable biphasic TCP dense ceramics composed of α-TCP and β-TCP were previously synthesized by Li and his co-workers [31]. They found that the ratio of α-TCP and β-TCP in the calcium phosphate particle was controlled by aging time and pH value during synthesis, but the degradation behavior of the biphasic TCP is unknown. Kamitakahara et al. implanted their α/ β-TCP porous ceramics, synthesized using additives of Mg, into bone defects made in rat tibias, and the results showed that dissolution rate of their α/β-TCP bioceramics was lower and higher than α-TCP and βTCP, respectively [32,33]. Despite the existence of a single study related to in vivo degradation of α/β-TCP ceramics performed by Kamitakahara et al., to our best knowledge, little work regarding in vitro degradation kinetics and bioactivity for porous dual α/β-TCP bioceramics in SBF is carefully reported and investigated. The co-existence of α- and β-TCP phase in biphasic TCP bioceramics is associated with the advantages of appropriate degradation rate of α-TCP and superb osteoinductivity/ biocompatibility derived from β-TCP. In the present study, hence, the porous α/β-TCP ceramic scaffolds were fabricated, and the preliminary in vitro dissolution behavior and bioactivity of porous biphasic TCP with variable α-TCP/β-TCP ratios under static and dynamic conditions in SBF were discussed. The results showcased that the porous α/β-TCP dual bioceramics displayed good in vitro bioactivity, and the adjustable degradation rate could be achieved by altering the ratio of each TCP phase in the materials, thereby, boding well application to bone tissue engineering. 2. Materials and methods 2.1. Materials Calcium hydroxide (Ca(OH)2, 99%) and phosphoric acid (H3PO4, 99%) were purchased from Chengdu Kelong Reagent Co., Ltd. (China).
Polyvinyl alcohol (PVA, (CH2CHOH)n, Mw = 10,000, 99%) was provided by Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Stearic acid particles (CH3(CH2)16COOH, Grade I, ≥ 98.5%) were supplied from Sigma-Aldrich (Shanghai, China). All other chemicals were of analytical reagent grade and were used as received unless noted. All aqueous solutions were prepared with de-ionized water (D.I. water).
2.2. Preparation of amorphous calcium phosphate (ACP) precursor The amorphous calcium phosphate (ACP) precursor powder was synthesized based on the established Ca(OH)2 and H3PO4 system. Briefly, Ca(OH)2 was dissolved in D.I. water at a concentration of 0.1 mol/L. Then, the suspension of Ca(OH)2 solution was added dropwise into the aqueous H3PO4 solution (0.133 mol/L) according to a Ca/P molar ratio of 1.50/1 with constant stirring at room temperature. The precipitation reaction occurred when kept at 5 °C for 6 h, and the pH value of supernatant was maintained in the range of 9–9.5 with ammonia. After reaction, the precipitate was filtered, rinsed with D.I. water, and finally dried under 105 °C. The precursor powders could be transformed to monophasic and biphasic TCPs by treating at 1000–1300 °C range without gas protection.
2.3. Preparation of porous TCP bioceramics 2.3.1. Porous green body Stearic acid particles were used as pore-forming agent to create porous bioceramics in the study. The high-purity spherical particles of stearic acid were first sieved into 100–400 mm. The selected stearic acid and ACP precursor powders were mixed at an appropriate ratio, and PVA solution (1% w/v) was incorporated as a binder. The mixed powders were then mold-shaped to the column samples with a diameter of 5 mm and a height of 15 mm under dry pressing condition, and the obtained samples were dried at 105 °C before sintering.
2.3.2. Sintering of porous α-TCP and β-TCP bioceramics The porous α-TCP and β-TCP bioceramics were prepared via a process of step-by-step heating/cooling approach as described in previous literature [15,34]: (1) Samples were placed in an oven and slowly heated to 300 °C for 0.5 h to remove the residual moisture, PVA and stearic acid; (2) Then, the temperature was raised to 900 °C and incubated for 0.5 h; (3) The temperature was elevated to 1180 °C for 2 h, and then cooled gradually to ambient temperature to obtain a porous βTCP bioceramic; (4) The temperature was raised rapidly to 1280 °C and kept for 2 h, and then cooled rapidly to room temperature resulting in the formation of porous α-TCP bioceramic. 2.4. Sintering of porous biphasic α/β-TCP bioceramics 2.4.1. α-TCP and β-TCP bioceramic powders The as-prepared ACP precursor powders were directly heated in the absence of pore-forming agent and mold-shaping. The heat treatment process of the pure α-TCP and β-TCP powders was the same as that of porous α-TCP and β-TCP bioceramics. 2.4.2. Porous biphasic α/β-TCP bioceramics The prepared α-TCP and β-TCP powders were mixed uniformly at a mass ratio of 80:20, 50:50 and 20:80, respectively. Subsequently, the mixtures with stearic acid were molded and sintered at 800 °C for 3 h to obtain the various porous biphasic α/β-TCP bioceramics. These porous α/β-TCPs were designated as α8/β2-TCP, α5/β5-TCP, and α2/β8TCP according to different α-TCP and β-TCP ratios, respectively, and the specific synthetic condition of each sample is listed in Table 1.
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015 Table 1 Composition and synthesizing condition of the porous biphasic α/β-TCP. Sample name
α-TCP phase (wt.%)
β-TCP phase (wt.%)
Sintering condition
α-TCP α8/β2-TCP α5/β5-TCP α2/β8-TCP β-TCP
100 80 50 20 0
0 20 50 80 100
1280 °C, quenching 800 °C 800 °C 800 °C 1180 °C, natural cooling
2.5. Characterization The crystalline phase of the TCP bioceramics was examined by X-ray diffraction analysis (XRD, D/max-A, Rigaku Corp., Tokyo, Japan) using a Cu target as radiation source (λ = 1.540598 Å). The diffraction angles (2θ) were set between 10° and 60°, with an incremental step size of 2 min−1. The phase identification was achieved by comparing the sample diffraction pattern with standard cards in ICDD-JCPDS database. Fourier transform infrared spectrometry (FTIR, Magna-IR 750, Nicolet, USA) was used to identify the functional groups of samples (KBr pellet). The spectra were recorded from 4000 cm−1 to 400 cm−1. In addition, the morphological structure of these dual α/β-TCP bioceramics was characterized by a field emission scanning electron microscope (FESEM, JSM-7500F, JEOL, Japan) at an accelerating voltage of 20 kV. All samples were coated by gold for 1 min before SEM observation. The porosity of the TCP bioceramics was measured by liquid displacement method. Six samples in each group were performed to provide an average and standard deviation. 2.6. Mechanical testing Disk samples of 1 cm diameter and 0.5 cm thickness were prepared for mechanical testing. The compressive strength of these porous TCP bioceramic scaffolds was evaluated using a single-grain compressive strength tester (TS-14, Chengdu, China) with a loading rate of 441 N/min. Six parallel specimens were used to provide an average and standard deviation. 2.7. In vitro dissolution behavior and bioactivity in SBF 2.7.1. In vitro static degradation experiment The 1 × SBF (Table S1) was prepared by dissolving the following chemicals in the sequence of NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, CaCl2, and Na2SO4 in distilled water and buffering to pH 7.4 with (CH2OH)3CNH2 and 1 M HCl at 37 °C. The in vitro degradation characteristic was evaluated in SBF as slow-release medium. Various porous TCP bioceramics (total weight 10 ± 0.05 g) were placed into clean beakers and incubated in 25 mL SBF for each sample. The beaker was then sealed and held in an incubator at a constant temperature of 37 °C. After immersion for scheduled times (0.5, 1, 3, 7 and 12 days), the samples were taken out, and the released Ca and P concentrations from the porous TCP bioceramics were tested.
1009
And the Ca2 + concentration released from various porous TCP bioceramics (CTCP) was Ctotal − CSBF, where CSBF represented the initial Ca concentration of SBF (=100 mg/L). The change of the phosphorus concentration was measured in the form of phosphomolybdate complex using a UV–visible spectrophotometer (Shimadzu, UV-2401PC, Japan), and the pH of the solution was monitored by a pH meter (Sartorius, pb-10, Germany) with an accuracy of ± 0.02. Six samples in each stage were analyzed to provide an average. 2.7.4. Bone-like apatite formation After 12 days of soaking in both static and dynamic SBFs, the microstructures of the TCP bioceramic samples were examined using the FESEM (JSM-7500F) after coating by gold. 2.7.5. Dissolution kinetic The dissolution rate of the porous TCP bioceramics under static and dynamic models was calculated using a calibration curve approach according to the Avrami equation for an ionic diffusion model. 3. Results and discussion 3.1. XRD and FT-IR results of TCP bioceramics In the present work, ACP precipitate powders with a Ca/P ratio of 1.50 were first wet-synthesized via reacting of Ca(OH)2 and H3PO4 under a basic ammonia condition. ACP consists of Ca9(PO4)6 units, and the units are agglomerated into larger particles with the help of polymeric molecules (PVA) binders. After the ACP is calcined with the removal of the binders, the units rearrange into an ordered structure through crystallization. The metastable α-TCP crystals from ACP rather than stable β-TCP are the first crystallized phase. However, different types of porous TCP ceramics could be obtained by sintering the ACP green body containing porogen at about 110–1300 °C for 3 h using a different cooling procedure. Quenching generates monophasic α-TCP, and natural cooling offers sufficient time for the transformation of α-TCP to thermodynamically stable β-TCP. To examine that TCP was the primary calcium phosphate phase and distinguish the differences between αTCP and β-TCP crystals, the XRD analysis was performed. As shown in Fig. 1, the peaks on the XRD patterns of the synthesized α-TCP and βTCP bioceramics closely matched the peaks of pure α-TCP (JCPD 090348) and β-TCP (JCPD 09-0169), respectively, indicating that all samples were predominantly TCP crystals. There was quite a difference in the XRD pattern of α-TCP and β-TCP bioceramics, where α-TCP revealed a main reflection at 2θ of 12.2°, 22.3°, 24.2°, and 30.9°, while the presence of β-TCP was confirmed by the main reflection at 27.8°, 25.9°,
2.7.2. In vitro dynamic degradation experiment These porous TCP bioceramics were placed in self-designed vessels with 25 mL SBF for each sample. A circulation flow system of SBF solution was created using a peristaltic pump at the rate of 30 mL/min. The system was also maintained at 37 °C in an incubator for ion release. Finally, the Ca and P element concentrations in the circulating fluid were determined using the same method. 2.7.3. Quantification of calcium and phosphorus concentrations The total calcium ion concentration of solution (Ctotal) in both static and dynamic degradation experiments was measured by a Hitachi 180/80 atomic absorption spectrophotometer (Hitachi, Tokyo, Japan).
Fig. 1. XRD patterns of the porous pure α-TCP, β-TCP, and three α/β-TCP biphasic bioceramics.
1010
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015
31.2° and 33.1° [35]. With respect to these porous α/β-TCP bioceramics, α-TCP and the β-TCP phases co-existed in the dual α8/β2-TCP, α5/β5TCP, and α2/β8-TCP. However, it was obvious that the relative intensity of α-TCP phase was reduced, combined with the increasing intensity of β-TCP phase, particularly the two most intense peaks at 30.8° (α-TCP) and 33.2° (β-TCP), with the decreasing α-TCP content from α8/β2-TCP to α2/β8-TCP, indicating distinct ratios of α-TCP and β-TCP content in three porous biphasic α/β-TCPs. In order to confirm the XRD results, FT-IR technology was well recommended and used to identify the functional groups of the obtained samples after calcination (Fig. 2). The typical broad peaks of the adsorbed H2O were observed at 3463 and 1638 cm−1. The bands at around 1027 and 1136 cm−1 were likely attributed to the triply degenerate ν3 antisymmetric P–O stretching modes, and the peak at 967 cm− 1 was assigned to ν1 non-degenerate symmetric P–O bond stretching band. Additionally, the peaks at about 608 and 549 cm− 1 belonged to the triply degenerate ν4 vibration of O–P–O bonds. Most of the time, it might become somewhat difficult to distinguish between the resemblant FT-IR spectra of α-TCP and β-TCP. The absorption bands of [PO4] functional groups confirmed the presence of TCP phase in all assynthesized samples. 3.2. Morphology and mechanical property of the porous biphasic α/β-TCP bioceramics As shown in Fig. 3, the SEM microstructure of the five porous TCP bioceramics revealed a significant difference between α-TCP and βTCP, with α-TCP displaying lack of round macropores, possibly due to quenching. However, pure β-TCP and α/β-TCP bioceramics all possessed uniformly interconnected pore structures of typical bone scaffold materials with sizes ranging from 200 to 500 μm. Besides, from the enlarged image, it could be found that the agglomerated TCP clusters of petal-like crystallites with about 2–5 μm micropores were present on the interior surface of the β-TCP-containing macropores (Fig. 3b, d). The porosity of these porous TCP bioceramics was similar with value of approximately 68% (Fig. S1) calculated by the liquid displacement method after being calcined. The macropore size and distribution of the pore could be tailored by the particle size and amount of stearic acid during the preparation. Previous work has proved that appropriate pore structure and size in the scaffold not only are helpful in ion exchange and absorption of functional proteins [36], such as the family of bone morphogenetic proteins (BMPs) which are the most potent growth factors in modulating osteo-differentiation of cells and enhancing bone formation [37], but also contribute to osteoblast proliferation [38], as well as promote bone ingrowth [39,40]. In addition to
Fig. 2. FT-IR spectra of the porous pure α-TCP, β-TCP, and three α/β-TCP biphasic bioceramics.
microstructure characterization and phase identification of the prepared porous bioceramics, their mechanical tests are also of great importance since the porous TCP may be used as biomaterial. Meanwhile, their mechanical properties may confirm the good sinter-ability of samples, due to necks or bonding formation. The compressive strength of the porous α-TCP, β-TCP, and three α/β-TCP bioceramics was shown in Fig. 4. Apparently, pure porous α-TCP and β-TCP possessed the lowest and highest compressive strengths (19.13 ± 0.25, and 22.11 ± 0.28 MPa, respectively) among these TCP bioceramics. Indeed, the compressive strength increased from 20.1 to 22.1 MPa for α/β-TCP samples with the increasing content of β-TCP phase in the α/β-TCP bioceramics, indicating that α/ β-TCP ceramics could have better mechanical property with higher βTCP content in them. Therefore, the biphasic α/β-TCP with abundant macropores and appropriate mechanical property would have a promising application as scaffold materials in bone tissue engineering. 3.3. In vitro static and dynamic dissolution behaviors TCP materials are degradable and reactive in human body fluids, and the degradation products (mainly Ca2+ and PO3− 4 ions) can make pivotal contributions to bone regeneration and repair. Consequently, TCP is one of the most accepted biomaterials for synthetic bone graft substitutes and cements in bone tissue engineering. The estimation of the released ion content of bioceramics in simulated body environment is an effective approach to investigate the dissolution characteristics of materials [41]. Variations of calcium and phosphorus concentration as a function of time in the SBF solution under static and dynamic conditions were presented in Figs. 5 and 6(a). The changes in calcium ion concentration included the increase by the TCP dissolution and the decrease by the apatite precipitation. The Ca2+ concentration of the testing solution increased with immersion time in all TCP groups. It implied that the TCP dissolution dominated the reaction in SBF before the Ca ionic concentration reached the apex during the 12 days of immersion. It was remarkable that the Ca and [P] elements released from α-TCP dominated throughout the test, and β-TCP displayed the lowest degradation rate. The order of dissolution rate for the five samples was as follows: α-TCP N α8/β2-TCP N α5/β5-TCP N α2/β8-TCP N β-TCP, indicating that higher α-TCP phase in the α/β-TCP contributed to increased degradation. In fact, bone is surrounded in tissue fluid, which accounts for about 15% of adult body weight, and it is constantly circulating in the body to maintain homeostasis. Thus, the implanted bone substitutes always remain in a dynamic equilibrium with the body fluids. In order to simulate tissue fluid dynamics and investigate the dynamic degradation of the porous TCP bioceramics, a peristaltic pump circulation device was used (Fig. 7). As shown in Fig. 5b, the curve of Ca2+ concentration in dynamic environment was similar to that in static SBF. However, different from static condition, within the first day Ca release increased abruptly, and slowed down after 7 days under dynamic environment. Similar phenomena were found in [P] concentration under both static and dynamic conditions as shown in Fig. 6(a). Besides, the evolution of the pH values in SBF vs. soaking time under both conditions of assay was also shown in Fig. 6(b). The pH values in SBF solution exhibited a slight decrease from about 7.38 to 7.21–7.28 until 12 days due to the partial depletion of OH− in solution for the formation of new apatite deposition. The degradation rate for the five TCPs also decreased in the order α-TCP N α8/β2-TCP N α5/β5-TCP N α2/β8-TCP N β-TCP, and the level of Ca concentration in α8/β2-TCP sample was about 1.11 and 1.33 times than that of α5/β5-TCP and α2/β8-TCP groups, respectively. The dissolution rate in dynamic condition was higher than that under static environment, consistent with previous studies suggesting that a dynamic environment significantly facilitated the degradation of biomaterials by constantly updating the ceramic surface [42]. It is well-known that the dissolution rate of a biomaterial depends on multiple aspects including microstructure, porosity and crystal phases. From the above discussion, the three porous α/β-TCP ceramics
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015
1011
Fig. 3. SEM images of the porous TCP hybrid bioceramics with different magnifications (×40; ×5000): (a) α-TCP, (b) β-TCP, (c) α8/β2-TCP, (d) α5/β5-TCP, (e) α2/β8-TCP.
had similar microstructure and porosity, therefore, it was suggested that the different constitution proportions of α and β TCP phases in materials could mainly contribute to the dissolution rates of α/β-TCP. The resorption of calcium phosphate biomaterials is proved to be beneficial to bone formation and free Ca2+ from calcium phosphate is considered as the origin of bioactivity. Some earlier studies, at the same time, conled to drastic alteration of firm that excessive dissolved Ca2+ and PO3− 4 the microenvironment combined with the deteriorated mechanical properties of calcium phosphate-based materials, disruption in the activity of host cells and creating adverse effects on adjacent tissues [43, 44]. Our porous α/β-TCP showed a steerable release of Ca2+ contents through altering the ratio of α and β TCP phases in the dual α/β-TCP bioceramics for optimizing degradation and osteoconductivity.
porous α5/β5-TCP bioceramic specimens after 12 days of immersion displayed a typical fine apatite microstructure under SEM, which was similar in composition and structure to bone apatite. It was observed that the crystals were poorly crystalline, consisting of plate-like crystals.
3.4. Bone-like apatite formation Since one of the significant characteristics of bioactive materials is their ability to bond with living bone through the formation of a bonelike apatite layer on their surfaces [45], the apatite formation on the porous dual α/β-TCP bioceramics after immersion in SBF under both static and dynamic conditions was assessed. As shown in Fig. 8, the
Fig. 4. Compressive strength of the porous α-TCP, β-TCP, and α/β-TCP biphasic bioceramics.
Fig. 5. The calcium ion concentrations degraded from various TCP bioceramics at different soaking time in static (a) and dynamic (b) SBF.
1012
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015
Fig. 6. Variation of phosphorous content (a) (after subtracting the initial [P] concentration of SBF) and pH value (b) with soaking time in SBF for TCP bioceramics under static and dynamic conditions.
Fig. 7. Schematic diagram of the dynamic degradation for the porous α/β-TCP bioceramics under dynamic SBF.
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015
1013
Fig. 8. SEM images of the porous α5/β5-TCP bioceramics acquired after soaking in static (a–b) and dynamic SBF (c–d) for 12 days with different magnifications.
From the high-resolution images of Fig 7b and d, the plates coalesced to form large apatite clusters with flower-like structure, and particles became interconnected on the surface of macropore in scaffolds. However, a continuous release of Ca and P could slow the reactivity of materials. The formation of the bone-like apatite layer may involve cation substitutions at the Ca2 + sites, and/or the substitution at PO34 − sites by ions, with the concomitant creation of calcium vacancies to satHPO2− 4 isfy the condition of electrical charge neutrality [46]. The SEM analysis revealed that the dissolution and precipitation process resulted in calcium phosphate globule formation, and the formation of a new apatite layer after immersion of the specimens in SBF confirmed that the porous α5/β5-TCP hybrid bioceramics had a good in vitro bioactivity, which has been thought to be one of the necessary conditions for biomedical applications. 3.5. Dissolution kinetics of the porous α/β-TCP biphasic bioceramics Next, another goal of the work is to present and calculate the dissolution kinetics of the porous dual α/β-TCP bioceramics to understand their degradation attributes. The dissolution kinetics of calcium phosphate can be described by a single or a mixture of the three following rate laws: linear, parabolic, and exponential. These three types of kinetics are explained by the following rate-determining mechanisms, respectively: (a) diffusion or adsorption, (b) surface spiral dissolution, and (c) surface nucleation [47]. In the present study, the dissolution of the porous biphasic α/β-TCP bioceramic is assumed to be diffusion controlled. On the bases of the dissolution results, the classic Avrami equation is put forward to describe the kinetics of the porous α/β-TCP bioceramics, and the kinetic model is expressed by: f ¼ 1 exp: −kt
n
where f is the dissolution rate of TCP bioceramics at different immersion time t, k is rate constant, and n represents the Avrami exponent which is a coefficient related to the material property and shape. This can be rewritten as: − ln ð1 f Þ ¼ kt
n
Furthermore, there are three types of dissolution kinetics model according to the value of “n” in the equation: (i) n b 1, the starting dissolution rate is infinite and the dissolution rate decreases with time, (ii) n = 1, the starting dissolution rate is finite, and (iii) n N 1, the starting dissolution rate is near zero, respectively. Obviously, the research model of the porous α/β-TCP hybrid bioceramics belonged to type (ii) with finite stating dissolution rate. The kinetic equation became y = −ln (1 − f ) = kt, and the logarithm of (1 − f ) is a linear function of time (t). In the current experimental conditions, the porous monophasic and biphasic TCP bioceramics gradually degraded in SBF until the concentration of calcium ion reached Cmax (the saturation value). Cmax values for α-TCP, α8/β2-TCP, α5/β5-TCP, α2/β8-TCP and βTCP were about 29.5, 28.3, 27.6, 26.7 and 26.1 mg/L after immersion in SBF for 30 days, respectively. Hence, f was calculated by CTCP/Cmax, in which CTCP represented the current concentration of calcium ion released from various porous TCP bioceramics. The resultant −ln (1 − f ) was plotted as a function of t as shown in Fig. 9, and the y value showed a good linear relation with immersion time (t) (with R2 value N 0.987), whose y-intercept approximated to zero, and slope represented k value (rate constant for five TCP bioceramics). The dissolution profile of the porous α/β-TCP was, thereby, well expressed by the equation −ln (1 − f ) = kt, and the rate constant (k) was calculated from the slope of curve displayed in Table S2. It could be found that the rate constant for the porous α/β-TCP increased with the contents of α-TCP phase. Namely, increased α-TCP levels
1014
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015
Acknowledgments This work was supported by the Open Fund of Sichuan Province Key Laboratory of Nonmetal Composite and Functional Materials (No. 11zxfk11).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.11.040.
References
Fig. 9. Linearization of the various porous TCP bioceramics dissolution under static (a) and dynamic (b) SBF using the Avrami equation y = −ln (1 − f) = kt.
promoted the degradation of α/β-TCP regardless of under static and dynamic environments, further confirming that the dissolution behavior of the porous biphasic complex could be steered depending on the components of α and β phases. Through fitting of the dissolution rate with the Avrami equation, the desired Ca2+ content, which is needed or sufficient for bone formation in the human body released from biphasic α/β-TCP at different time points, could be predicted, providing a help to design the porous α/β-TCP bioceramics for bone tissue engineering. 4. Conclusions Overall, the ACP-derived porous TCP hybrid bioceramics displayed a homogeneous microstructural character of interconnected macropore structure and the co-existence of α-TCP and β-TCP phases in each α/ β-TCP. Bioactivity and dissolution behaviors in SBF were conducted by immersing the scaffold specimens for 12 days. The porous α/β-TCP scaffolds showed good in vitro bioactivity due to the formation of bone-like apatite phase on the surface. The dissolution results showed that dynamic environment enhanced the dissolution of α/β-TCP than that under static condition. The porous biphasic α/β-TCP bioceramics with adjustable degradation rate were obtained via tuning the proportion of α- and β-TCP content, and the dissolution rate of porous TCP biomaterials also could be well-expressed and fitted using the Avrami formula (−ln (1 − f ) = kt) for an ionic diffusion model. Due to excellent bioactivity and controlled degradability, therefore, the porous biphasic α/β-TCP bioceramics could offer promising potential for the repair and replacement of bone.
[1] R. Langer, D.A. Tirrell, Designing materials for biology and medicine, Nature 428 (2004) 487–492. [2] A.K. Gaharwar, S.M. Mihaila, A. Swami, A. Patel, S. Sant, R.L. Reis, A.P. Marques, M.E. Gomes, A. Khademhosseini, Bioactive silicate nanoplatelets for osteogenic differentiation of human mesenchymal stem cells, Adv. Mater. 25 (2013) 3329–3336. [3] R.R. Betz, Limitations of autograft and allograft: new synthetic solutions, Orthopedics 25 (2002) s561–s570. [4] B.M. Holzapfel, J.C. Reichert, J.-T. Schantz, U. Gbureck, L. Rackwitz, U. Noeth, F. Jakob, M. Rudert, J. Groll, D.W. Hutmacher, How smart do biomaterials need to be? A translational science and clinical point of view, Adv. Drug Deliv. Rev. 65 (2013) 581–603. [5] S. Higashi, T. Yamamuro, T. Nakamura, Y. Ikada, S.-H. Hyon, K. Jamshidi, Polymerhydroxyapatite composites for biodegradable bone fillers, Biomaterials 7 (1986) 183–187. [6] E. Willbold, X. Gu, D. Albert, K. Kalla, K. Bobe, M. Brauneis, C. Janning, J. Nellesen, W. Czayka, W. Tillmann, Effect of the addition of low rare earth elements (lanthanum, neodymium, cerium) on the biodegradation and biocompatibility of magnesium, Acta Biomater. 11 (2015) 554–562. [7] W. Suchanek, M. Yoshimura, Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants, J. Mater. Res. 13 (1998) 94–117. [8] U. Ripamonti, L.C. Roden, L.F. Renton, Osteoinductive hydroxyapatite-coated titanium implants, Biomaterials 33 (2012) 3813–3823. [9] B. Liu, D.X. Lun, Current application of β-tricalcium phosphate composites in orthopaedics, Orthop. Surg. 4 (2012) 139–144. [10] H.-S. Ryu, K.S. Hong, J.-K. Lee, D.J. Kim, J.H. Lee, B.-S. Chang, D.-h. Lee, C.-K. Lee, S.-S. Chung, Magnesia-doped HA/β-TCP ceramics and evaluation of their biocompatibility, Biomaterials 25 (2004) 393–401. [11] M. Yashima, A. Sakai, High-temperature neutron powder diffraction study of the structural phase transition between α and α′ phases in tricalcium phosphate Ca3(PO4)2, Chem. Phys. Lett. 372 (2003) 779–783. [12] X. Wei, O. Ugurlu, A. Ankit, H.Y. Acar, M. Akinc, Dissolution behavior of Si, Zncodoped tricalcium phosphates, Mater. Sci. Eng. C Mater. 29 (2009) 126–135. [13] N. Matsumoto, K. Yoshida, K. Hashimoto, Y. Toda, Thermal stability of β-tricalcium phosphate doped with monovalent metal ions, Mater. Res. Bull. 44 (2009) 1889–1894. [14] I. Martinez, P. Velasquez, L. Meseguer-Olmo, A. Bernabeu-Esclapez, P. De Aza, Preparation and characterization of novel bioactive α-tricalcium phosphate doped with dicalcium silicate ceramics, Mater. Sci. Eng. C Mater. 32 (2012) 878–886. [15] H. Yuan, J. De Bruijn, Y. Li, J. Feng, Z. Yang, K. De Groot, X. Zhang, Bone formation induced by calcium phosphate ceramics in soft tissue of dogs: a comparative study between porous α-TCP and β-TCP, J. Mater. Sci. Mater. Med. 12 (2001) 7–13. [16] J. Lu, M. Descamps, J. Dejou, G. Koubi, P. Hardouin, J. Lemaitre, J.-P. Proust, The biodegradation mechanism of calcium phosphate biomaterials in bone, J. Biomed. Mater. Res. 63 (2002) 408–412. [17] N. Kondo, A. Ogose, K. Tokunaga, H. Umezu, K. Arai, N. Kudo, M. Hoshino, H. Inoue, H. Irie, K. Kuroda, Osteoinduction with highly purified β-tricalcium phosphate in dog dorsal muscles and the proliferation of osteoclasts before heterotopic bone formation, Biomaterials 27 (2006) 4419–4427. [18] A. Ogose, N. Kondo, H. Umezu, T. Hotta, H. Kawashima, K. Tokunaga, T. Ito, N. Kudo, M. Hoshino, W. Gu, Histological assessment in grafts of highly purified betatricalcium phosphate (OSferion®) in human bones, Biomaterials 27 (2006) 1542–1549. [19] J. Huang, E. Ten, G. Liu, M. Finzen, W. Yu, J.S. Lee, E. Saiz, A.P. Tomsia, Biocomposites of pHEMA with HA/β-TCP (60/40) for bone tissue engineering: swelling, hydrolytic degradation, and in vitro behavior, Polymer 54 (2013) 1197–1207. [20] X. Xiao, R. Liu, Q. Huang, X. Ding, Preparation and characterization of hydroxyapatite/ polycaprolactone–chitosan composites, J. Mater. Sci. Mater. Med. 20 (2009) 2375–2383. [21] N. Sultana, T.H. Khan, In vitro degradation of PHBV scaffolds and nHA/PHBV composite scaffolds containing hydroxyapatite nanoparticles for bone tissue engineering, J. Nanomater. 2012 (2012) 1. [22] L. Mao, G. Yuan, J. Niu, Y. Zong, W. Ding, In vitro degradation behavior and biocompatibility of Mg–Nd–Zn–Zr alloy by hydrofluoric acid treatment, Mater. Sci. Eng. C 33 (2013) 242–250. [23] K. Hagihara, S. Shakudo, K. Fujii, T. Nakano, Degradation behavior of Ca–Mg–Zn intermetallic compounds for use as biodegradable implant materials, Mater. Sci. Eng. C 44 (2014) 285–292.
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015 [24] M. Houmard, Q. Fu, M. Genet, E. Saiz, A.P. Tomsia, On the structural, mechanical, and biodegradation properties of HA/β-TCP robocast scaffolds, J. Biomed. Mater. Res. B. Appl. Biomater. 101 (2013) 1233–1242. [25] C. Shuai, J. Zhuang, H. Hu, S. Peng, D. Liu, J. Liu, In vitro bioactivity and degradability of β-tricalcium phosphate porous scaffold fabricated via selective laser sintering, Biotechnol. Appl. Biochem. 60 (2013) 266–273. [26] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 27 (2006) 2907–2915. [27] Y. Yang, Y. Zhao, G. Tang, H. Li, X. Yuan, Y. Fan, In vitro degradation of porous poly (Llactide-co-glycolide)/β-tricalcium phosphate (PLGA/β-TCP) scaffolds under dynamic and static conditions, Polym. Degrad. Stab. 93 (2008) 1838–1845. [28] J.-p. Seo, N. Tsuzuki, S. Haneda, K. Yamada, H. Furuoka, Y. Tabata, N. Sasaki, Proliferation of equine bone marrow-derived mesenchymal stem cells in gelatin/βtricalcium phosphate sponges, Res. Vet. Sci. 93 (2012) 1481–1486. [29] S. Kobayashi, S. Yamadi, Strain rate dependency of mechanical properties of TCP/ PLLA composites after immersion in simulated body environments, Compos. Sci. Technol. 70 (2010) 1820–1825. [30] S.-H. Kwon, Y.-K. Jun, S.-H. Hong, H.-E. Kim, Synthesis and dissolution behavior of βTCP and HA/β-TCP composite powders, J. Eur. Ceram. Soc. 23 (2003) 1039–1045. [31] Y. Li, W. Weng, K.C. Tam, Novel highly biodegradable biphasic tricalcium phosphates composed of α-tricalcium phosphate and β-tricalcium phosphate, Acta Biomater. 3 (2007) 251–254. [32] M. Kamitakahara, C. Ohtsuki, T. Miyazaki, Review paper: behavior of ceramic biomaterials derived from tricalcium phosphate in physiological condition, J. Biomater. Appl. 23 (2008) 197–212. [33] M. Kamitakahara, C. Ohtsuki, M. Oishi, S. Ogata, T. Miyazaki, M. Tanihara, Preparation of porous biphasic tricalcium phosphate and its in vivo behavior, Key Eng. Mater. 281-284 (2005). [34] R. LeGeros, S. Lin, R. Rohanizadeh, D. Mijares, J. LeGeros, Biphasic calcium phosphate bioceramics: preparation, properties and applications, J. Mater. Sci. Mater. Med. 14 (2003) 201–209. [35] K. Sariibrahimoglu, J.G. Wolke, S.C. Leeuwenburgh, L. Yubao, J.A. Jansen, Injectable biphasic calcium phosphate cements as a potential bone substitute, J. Biomed. Mater. Res. B. Appl. Biomater. 102 (2014) 415–422.
1015
[36] J. Jansen, J. Vehof, P. Ruhe, H. Kroeze-Deutman, Y. Kuboki, H. Takita, E. Hedberg, A. Mikos, Growth factor-loaded scaffolds for bone engineering, J. Control. Release 101 (2005) 127–136. [37] G.B. Bishop, T.A. Einhorn, Current and future clinical applications of bone morphogenetic proteins in orthopaedic trauma surgery, Int. Orthop. 31 (2007) 721–727. [38] J. Zeltinger, J.K. Sherwood, D.A. Graham, R. Müeller, L.G. Griffith, Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition, Tissue Eng. 7 (2001) 557–572. [39] O. Gauthier, J.-M. Bouler, E. Aguado, P. Pilet, G. Daculsi, Macroporous biphasic calcium phosphate ceramics: influence of macropore diameter and macroporosity percentage on bone ingrowth, Biomaterials 19 (1998) 133–139. [40] H. Kiba, N. Kuboyama, R. Uchida, T. Ishizaki, N. Nishiyama, Bone ingrowth into the parallel cylindrical tubes with different sizes of porous hydroxyapatite implanted into the rabbits, J. Hard Tissue Biol. 21 (2012) 307–314. [41] Q. Wang, Q. Wang, J. Wang, X. Zhang, X. Yu, C. Wan, Degradation kinetics of calcium polyphosphate bioceramic: an experimental and theoretical study, Mater. Res. 12 (2009) 495–501. [42] C. Agrawal, J. McKinney, D. Lanctot, K. Athanasiou, Effects of fluid flow on the in vitro degradation kinetics of biodegradable scaffolds for tissue engineering, Biomaterials 21 (2000) 2443–2452. [43] T. Suzuki, T. Yamamoto, M. Toriyama, K. Nishizawa, Y. Yokogawa, M.R. Mucalo, Y. Kawamoto, F. Nagata, T. Kameyama, Surface instability of calcium phosphate ceramics in tissue culture medium and the effect on adhesion and growth of anchorage-dependent animal cells, J. Biomed. Mater. Res. 34 (1997) 507–517. [44] S. Yamada, D. Heymann, J.-M. Bouler, G. Daculsi, Osteoclastic resorption of calcium phosphate ceramics with different hydroxyapatite/β-tricalcium phosphate ratios, Biomaterials 18 (1997) 1037–1041. [45] T. Kokubo, H.M. Kim, M. Kawashita, Novel bioactive materials with different mechanical properties, Biomaterials 24 (2003) 2161–2175. [46] Y.F. Chou, W.A. Chiou, Y. Xu, J.C. Dunn, B.M. Wu, The effect of pH on the structural evolution of accelerated biomimetic apatite, Biomaterials 25 (2004) 5323–5331. [47] R. Tang, W. Wu, M. Haas, G.H. Kinetics of dissolution of β-tricalcium phosphate, Langmuir 17 (2001) 3480–3485.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Preparation, characterization and in vitro dissolution behavior of porous biphasic α/β-tricalcium phosphate bioceramics Lu Xie a, Haiyang Yu a,⁎, Yi Deng b,c,⁎⁎, Weizhong Yang b, Li Liao b, Qin Long b a b c
State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University, Chengdu 610065, China School of Chemical Engineering, Sichuan University, Chengdu 610065, China Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
a r t i c l e
i n f o
Article history: Received 13 August 2015 Received in revised form 28 October 2015 Accepted 13 November 2015 Available online 14 November 2015 Keywords: Porous α/β-tricalcium phosphate Bioceramic Dissolution Bone tissue engineering
a b s t r a c t The ideal bone tissue engineering scaffolds are long-cherished with the properties of interconnected macroporous structures, adjustable degradation and excellent biocompatibility. Here, a series of porous α/βtricalcium phosphate (α/β-TCP) biphasic bioceramics with different phase ratios of α-TCP and β-TCP were successfully synthesized by heating an amorphous calcium phosphate precursor. The chemical and morphological characterization showed that α- and β-TCP phases co-existed in the α/β-TCP bioceramics and they had interconnected pore structures with size between 200 and 500 μm. The in vitro dissolution behavior and bioactivity of the dual α/β-TCP were also probed in static and dynamic SBF for the first time. The results revealed that α/β-TCP scaffolds had good in vitro bioactivity, as the formation of bone-like apatite layers was induced on the scaffolds after mineralization in SBF. Moreover, dissolution rate of α/β-TCP bioceramics in dynamic environment was higher than that under static condition. Compared with monophasic TCP ceramics, these porous α/β-TCP bioceramics displayed a tailored dissolution rate proportionate to the TCP content (α and β) in the materials. Further, the degradation profile of porous α/β-TCP was well-described by Avrami equation. The porous dual α/β-TCP bioceramics with controllable degradation behavior hold great potential to be applied in bone tissue engineering as bone substitutes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The incidence of bone deficiencies associated with osteoporosis, trauma, congenital malformations, and surgical resections is rising substantially so that governments have to continuously escalate budget to balance the growing healthcare expenditures every year [1,2]. Current treatments of large bone defects, which rely on the use of autologous grafts (the “gold standard” of bone substitution) and allograft transplantation [3], have limited clinical potential due to their inherent limitations, such as donor site morbidity, available quantities, and host immune rejection. These imperfections have already led to synthetic bone grafts becoming a powerful alternative in replacing autologous grafts. On the other hand, one of the actual and much-needed demands in orthopedics is the clinical availability of degradable implants [4–6]. In some clinical applications, such as fracture treatment, permanent metal or polymer implants are not necessary or even disadvantageous, and a
⁎ Correspondence to: H. Yu, State Key Laboratory of Oral Diseases, Sichuan University, No. 24 South Section 1, Yihuan Road, Chengdu 610065, China. ⁎⁎ Correspondence to: Yi Deng, Academy for Advanced Interdisciplinary Studies, Peking University, No. 5 Yiheyuan Road, Haidian District, Beijing, 100871, China. E-mail addresses: [email protected] (H. Yu), [email protected] (Y. Deng).
http://dx.doi.org/10.1016/j.msec.2015.11.040 0928-4931/© 2015 Elsevier B.V. All rights reserved.
temporary implant would be much more suitable. Temporary implants made of biodegradable materials are destined to dissolve and degrade postoperatively synchronized with bone healing periods, so that a second surgery for implant removal is escapable, obviating surgery and associated cost, as well as unnecessary health risks and pain for patients. Thus, the design and construction of controllable degradable materials in the body environment is still an imperative and hot research area in the application of bone tissue engineering. Tricalcium phosphate (Ca3(PO4)2, TCP), one member of the calcium phosphate ceramics family, has been considered as an ideal temporary scaffold used for bone/dental repair replacing autogenous bone, because of its close chemical resemblance to biological apatite present in human hard tissues [7–9]. TCP has four polymorphic forms [10]: α, β, α′ and γ. β-tricalcium phosphate (β-TCP) is stable at room temperature and reconstructively transforms into α-tricalcium phosphate (α-TCP) between 1185 and 1430 °C, which is metastably retained during the cooling until room temperature; α′- and γ-TCP form above 1430 °C and under high pressures, respectively, but they lack interest, since it is difficult for them to attain a single phase owing to their metastability [11]. Nevertheless, α-TCP has restricted biomedical applications and is mainly employed in bone cement because of its high resorbability and reactivity [12,13]. It is reported that the solubility and/or degradation rate of α-TCP are much higher than those of β-TCP [14]. In body fluids,
1008
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015
the higher local Ca2+ and PO3− concentration caused by the rapid dis4 solution of α-TCP bioceramic might hamper the bone formation [15]. In contrast, β-TCP is famous for its remarkable osteoconductivity and biocompatibility. Lu et al. previously studied the degradation mechanism of β-TCP in bone and found a new lamellar bone formation following direct contact with the β-TCP implant and surrounding osteoblasts after a few weeks of implantation [16]. It was progressively absorbed in vivo by a cell-mediated process and replaced by new bone, reported by Kondo et al. [17] and Ogose et al. [18]. However, β-TCP also presents poor reactivity as a pure material, because it suffers from drawbacks such as slow in vivo bone formation and less-than-ideal degradation/ solubility. Furthermore, the difficulty in matching the degradation and the growth rates of new bone is a tough challenge for the clinical application of TCP bioceramics, because the degradation rate of a single calcium phosphate phase cannot be modulated. The degradation rate would be broadened if the two polymorphs of the TCP ceramics mentioned above are combined. Complete biodegradation within 4–6 months is required for biomaterials [19] in clinical trials of bone regeneration and repair. The degradation rate of the materials should be regulated to conform to the clinical demands, therefore, the investigation of degradability and understanding of the dissolution kinetic of TCP biomaterials remain desired. Generally, in vitro degradation performance of materials is often studied in physiological saline [20], phosphate buffered saline (PBS) solution [21,22], Hanks' balanced salt solution [23], and simulated body fluid (SBF) [24,25]. Among these solutions, the chemical composition and ion concentration of SBF are nearly equal to that of human blood plasma, so SBF is extensively applied in assessment for the dissolution behavior and in vitro bioactivity of a biomaterial [26]. Currently, although there is great progress in the preparation of a variety of βTCP-based biocomposites including PLGA/β-TCP [27], gelatin/β-TCP [28], PLLA/β-TCP composite [29], and HA/β-TCP biphasic calcium phosphate (called BCP) [30] with the goal of modulating the degradation rate, the degradation rate range of these β-TCP-based composites still cannot satisfy the clinical standards, and it is a challenge to synthesize highly degradable calcium phosphates. New degradable biphasic TCP dense ceramics composed of α-TCP and β-TCP were previously synthesized by Li and his co-workers [31]. They found that the ratio of α-TCP and β-TCP in the calcium phosphate particle was controlled by aging time and pH value during synthesis, but the degradation behavior of the biphasic TCP is unknown. Kamitakahara et al. implanted their α/ β-TCP porous ceramics, synthesized using additives of Mg, into bone defects made in rat tibias, and the results showed that dissolution rate of their α/β-TCP bioceramics was lower and higher than α-TCP and βTCP, respectively [32,33]. Despite the existence of a single study related to in vivo degradation of α/β-TCP ceramics performed by Kamitakahara et al., to our best knowledge, little work regarding in vitro degradation kinetics and bioactivity for porous dual α/β-TCP bioceramics in SBF is carefully reported and investigated. The co-existence of α- and β-TCP phase in biphasic TCP bioceramics is associated with the advantages of appropriate degradation rate of α-TCP and superb osteoinductivity/ biocompatibility derived from β-TCP. In the present study, hence, the porous α/β-TCP ceramic scaffolds were fabricated, and the preliminary in vitro dissolution behavior and bioactivity of porous biphasic TCP with variable α-TCP/β-TCP ratios under static and dynamic conditions in SBF were discussed. The results showcased that the porous α/β-TCP dual bioceramics displayed good in vitro bioactivity, and the adjustable degradation rate could be achieved by altering the ratio of each TCP phase in the materials, thereby, boding well application to bone tissue engineering. 2. Materials and methods 2.1. Materials Calcium hydroxide (Ca(OH)2, 99%) and phosphoric acid (H3PO4, 99%) were purchased from Chengdu Kelong Reagent Co., Ltd. (China).
Polyvinyl alcohol (PVA, (CH2CHOH)n, Mw = 10,000, 99%) was provided by Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Stearic acid particles (CH3(CH2)16COOH, Grade I, ≥ 98.5%) were supplied from Sigma-Aldrich (Shanghai, China). All other chemicals were of analytical reagent grade and were used as received unless noted. All aqueous solutions were prepared with de-ionized water (D.I. water).
2.2. Preparation of amorphous calcium phosphate (ACP) precursor The amorphous calcium phosphate (ACP) precursor powder was synthesized based on the established Ca(OH)2 and H3PO4 system. Briefly, Ca(OH)2 was dissolved in D.I. water at a concentration of 0.1 mol/L. Then, the suspension of Ca(OH)2 solution was added dropwise into the aqueous H3PO4 solution (0.133 mol/L) according to a Ca/P molar ratio of 1.50/1 with constant stirring at room temperature. The precipitation reaction occurred when kept at 5 °C for 6 h, and the pH value of supernatant was maintained in the range of 9–9.5 with ammonia. After reaction, the precipitate was filtered, rinsed with D.I. water, and finally dried under 105 °C. The precursor powders could be transformed to monophasic and biphasic TCPs by treating at 1000–1300 °C range without gas protection.
2.3. Preparation of porous TCP bioceramics 2.3.1. Porous green body Stearic acid particles were used as pore-forming agent to create porous bioceramics in the study. The high-purity spherical particles of stearic acid were first sieved into 100–400 mm. The selected stearic acid and ACP precursor powders were mixed at an appropriate ratio, and PVA solution (1% w/v) was incorporated as a binder. The mixed powders were then mold-shaped to the column samples with a diameter of 5 mm and a height of 15 mm under dry pressing condition, and the obtained samples were dried at 105 °C before sintering.
2.3.2. Sintering of porous α-TCP and β-TCP bioceramics The porous α-TCP and β-TCP bioceramics were prepared via a process of step-by-step heating/cooling approach as described in previous literature [15,34]: (1) Samples were placed in an oven and slowly heated to 300 °C for 0.5 h to remove the residual moisture, PVA and stearic acid; (2) Then, the temperature was raised to 900 °C and incubated for 0.5 h; (3) The temperature was elevated to 1180 °C for 2 h, and then cooled gradually to ambient temperature to obtain a porous βTCP bioceramic; (4) The temperature was raised rapidly to 1280 °C and kept for 2 h, and then cooled rapidly to room temperature resulting in the formation of porous α-TCP bioceramic. 2.4. Sintering of porous biphasic α/β-TCP bioceramics 2.4.1. α-TCP and β-TCP bioceramic powders The as-prepared ACP precursor powders were directly heated in the absence of pore-forming agent and mold-shaping. The heat treatment process of the pure α-TCP and β-TCP powders was the same as that of porous α-TCP and β-TCP bioceramics. 2.4.2. Porous biphasic α/β-TCP bioceramics The prepared α-TCP and β-TCP powders were mixed uniformly at a mass ratio of 80:20, 50:50 and 20:80, respectively. Subsequently, the mixtures with stearic acid were molded and sintered at 800 °C for 3 h to obtain the various porous biphasic α/β-TCP bioceramics. These porous α/β-TCPs were designated as α8/β2-TCP, α5/β5-TCP, and α2/β8TCP according to different α-TCP and β-TCP ratios, respectively, and the specific synthetic condition of each sample is listed in Table 1.
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015 Table 1 Composition and synthesizing condition of the porous biphasic α/β-TCP. Sample name
α-TCP phase (wt.%)
β-TCP phase (wt.%)
Sintering condition
α-TCP α8/β2-TCP α5/β5-TCP α2/β8-TCP β-TCP
100 80 50 20 0
0 20 50 80 100
1280 °C, quenching 800 °C 800 °C 800 °C 1180 °C, natural cooling
2.5. Characterization The crystalline phase of the TCP bioceramics was examined by X-ray diffraction analysis (XRD, D/max-A, Rigaku Corp., Tokyo, Japan) using a Cu target as radiation source (λ = 1.540598 Å). The diffraction angles (2θ) were set between 10° and 60°, with an incremental step size of 2 min−1. The phase identification was achieved by comparing the sample diffraction pattern with standard cards in ICDD-JCPDS database. Fourier transform infrared spectrometry (FTIR, Magna-IR 750, Nicolet, USA) was used to identify the functional groups of samples (KBr pellet). The spectra were recorded from 4000 cm−1 to 400 cm−1. In addition, the morphological structure of these dual α/β-TCP bioceramics was characterized by a field emission scanning electron microscope (FESEM, JSM-7500F, JEOL, Japan) at an accelerating voltage of 20 kV. All samples were coated by gold for 1 min before SEM observation. The porosity of the TCP bioceramics was measured by liquid displacement method. Six samples in each group were performed to provide an average and standard deviation. 2.6. Mechanical testing Disk samples of 1 cm diameter and 0.5 cm thickness were prepared for mechanical testing. The compressive strength of these porous TCP bioceramic scaffolds was evaluated using a single-grain compressive strength tester (TS-14, Chengdu, China) with a loading rate of 441 N/min. Six parallel specimens were used to provide an average and standard deviation. 2.7. In vitro dissolution behavior and bioactivity in SBF 2.7.1. In vitro static degradation experiment The 1 × SBF (Table S1) was prepared by dissolving the following chemicals in the sequence of NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, CaCl2, and Na2SO4 in distilled water and buffering to pH 7.4 with (CH2OH)3CNH2 and 1 M HCl at 37 °C. The in vitro degradation characteristic was evaluated in SBF as slow-release medium. Various porous TCP bioceramics (total weight 10 ± 0.05 g) were placed into clean beakers and incubated in 25 mL SBF for each sample. The beaker was then sealed and held in an incubator at a constant temperature of 37 °C. After immersion for scheduled times (0.5, 1, 3, 7 and 12 days), the samples were taken out, and the released Ca and P concentrations from the porous TCP bioceramics were tested.
1009
And the Ca2 + concentration released from various porous TCP bioceramics (CTCP) was Ctotal − CSBF, where CSBF represented the initial Ca concentration of SBF (=100 mg/L). The change of the phosphorus concentration was measured in the form of phosphomolybdate complex using a UV–visible spectrophotometer (Shimadzu, UV-2401PC, Japan), and the pH of the solution was monitored by a pH meter (Sartorius, pb-10, Germany) with an accuracy of ± 0.02. Six samples in each stage were analyzed to provide an average. 2.7.4. Bone-like apatite formation After 12 days of soaking in both static and dynamic SBFs, the microstructures of the TCP bioceramic samples were examined using the FESEM (JSM-7500F) after coating by gold. 2.7.5. Dissolution kinetic The dissolution rate of the porous TCP bioceramics under static and dynamic models was calculated using a calibration curve approach according to the Avrami equation for an ionic diffusion model. 3. Results and discussion 3.1. XRD and FT-IR results of TCP bioceramics In the present work, ACP precipitate powders with a Ca/P ratio of 1.50 were first wet-synthesized via reacting of Ca(OH)2 and H3PO4 under a basic ammonia condition. ACP consists of Ca9(PO4)6 units, and the units are agglomerated into larger particles with the help of polymeric molecules (PVA) binders. After the ACP is calcined with the removal of the binders, the units rearrange into an ordered structure through crystallization. The metastable α-TCP crystals from ACP rather than stable β-TCP are the first crystallized phase. However, different types of porous TCP ceramics could be obtained by sintering the ACP green body containing porogen at about 110–1300 °C for 3 h using a different cooling procedure. Quenching generates monophasic α-TCP, and natural cooling offers sufficient time for the transformation of α-TCP to thermodynamically stable β-TCP. To examine that TCP was the primary calcium phosphate phase and distinguish the differences between αTCP and β-TCP crystals, the XRD analysis was performed. As shown in Fig. 1, the peaks on the XRD patterns of the synthesized α-TCP and βTCP bioceramics closely matched the peaks of pure α-TCP (JCPD 090348) and β-TCP (JCPD 09-0169), respectively, indicating that all samples were predominantly TCP crystals. There was quite a difference in the XRD pattern of α-TCP and β-TCP bioceramics, where α-TCP revealed a main reflection at 2θ of 12.2°, 22.3°, 24.2°, and 30.9°, while the presence of β-TCP was confirmed by the main reflection at 27.8°, 25.9°,
2.7.2. In vitro dynamic degradation experiment These porous TCP bioceramics were placed in self-designed vessels with 25 mL SBF for each sample. A circulation flow system of SBF solution was created using a peristaltic pump at the rate of 30 mL/min. The system was also maintained at 37 °C in an incubator for ion release. Finally, the Ca and P element concentrations in the circulating fluid were determined using the same method. 2.7.3. Quantification of calcium and phosphorus concentrations The total calcium ion concentration of solution (Ctotal) in both static and dynamic degradation experiments was measured by a Hitachi 180/80 atomic absorption spectrophotometer (Hitachi, Tokyo, Japan).
Fig. 1. XRD patterns of the porous pure α-TCP, β-TCP, and three α/β-TCP biphasic bioceramics.
1010
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015
31.2° and 33.1° [35]. With respect to these porous α/β-TCP bioceramics, α-TCP and the β-TCP phases co-existed in the dual α8/β2-TCP, α5/β5TCP, and α2/β8-TCP. However, it was obvious that the relative intensity of α-TCP phase was reduced, combined with the increasing intensity of β-TCP phase, particularly the two most intense peaks at 30.8° (α-TCP) and 33.2° (β-TCP), with the decreasing α-TCP content from α8/β2-TCP to α2/β8-TCP, indicating distinct ratios of α-TCP and β-TCP content in three porous biphasic α/β-TCPs. In order to confirm the XRD results, FT-IR technology was well recommended and used to identify the functional groups of the obtained samples after calcination (Fig. 2). The typical broad peaks of the adsorbed H2O were observed at 3463 and 1638 cm−1. The bands at around 1027 and 1136 cm−1 were likely attributed to the triply degenerate ν3 antisymmetric P–O stretching modes, and the peak at 967 cm− 1 was assigned to ν1 non-degenerate symmetric P–O bond stretching band. Additionally, the peaks at about 608 and 549 cm− 1 belonged to the triply degenerate ν4 vibration of O–P–O bonds. Most of the time, it might become somewhat difficult to distinguish between the resemblant FT-IR spectra of α-TCP and β-TCP. The absorption bands of [PO4] functional groups confirmed the presence of TCP phase in all assynthesized samples. 3.2. Morphology and mechanical property of the porous biphasic α/β-TCP bioceramics As shown in Fig. 3, the SEM microstructure of the five porous TCP bioceramics revealed a significant difference between α-TCP and βTCP, with α-TCP displaying lack of round macropores, possibly due to quenching. However, pure β-TCP and α/β-TCP bioceramics all possessed uniformly interconnected pore structures of typical bone scaffold materials with sizes ranging from 200 to 500 μm. Besides, from the enlarged image, it could be found that the agglomerated TCP clusters of petal-like crystallites with about 2–5 μm micropores were present on the interior surface of the β-TCP-containing macropores (Fig. 3b, d). The porosity of these porous TCP bioceramics was similar with value of approximately 68% (Fig. S1) calculated by the liquid displacement method after being calcined. The macropore size and distribution of the pore could be tailored by the particle size and amount of stearic acid during the preparation. Previous work has proved that appropriate pore structure and size in the scaffold not only are helpful in ion exchange and absorption of functional proteins [36], such as the family of bone morphogenetic proteins (BMPs) which are the most potent growth factors in modulating osteo-differentiation of cells and enhancing bone formation [37], but also contribute to osteoblast proliferation [38], as well as promote bone ingrowth [39,40]. In addition to
Fig. 2. FT-IR spectra of the porous pure α-TCP, β-TCP, and three α/β-TCP biphasic bioceramics.
microstructure characterization and phase identification of the prepared porous bioceramics, their mechanical tests are also of great importance since the porous TCP may be used as biomaterial. Meanwhile, their mechanical properties may confirm the good sinter-ability of samples, due to necks or bonding formation. The compressive strength of the porous α-TCP, β-TCP, and three α/β-TCP bioceramics was shown in Fig. 4. Apparently, pure porous α-TCP and β-TCP possessed the lowest and highest compressive strengths (19.13 ± 0.25, and 22.11 ± 0.28 MPa, respectively) among these TCP bioceramics. Indeed, the compressive strength increased from 20.1 to 22.1 MPa for α/β-TCP samples with the increasing content of β-TCP phase in the α/β-TCP bioceramics, indicating that α/ β-TCP ceramics could have better mechanical property with higher βTCP content in them. Therefore, the biphasic α/β-TCP with abundant macropores and appropriate mechanical property would have a promising application as scaffold materials in bone tissue engineering. 3.3. In vitro static and dynamic dissolution behaviors TCP materials are degradable and reactive in human body fluids, and the degradation products (mainly Ca2+ and PO3− 4 ions) can make pivotal contributions to bone regeneration and repair. Consequently, TCP is one of the most accepted biomaterials for synthetic bone graft substitutes and cements in bone tissue engineering. The estimation of the released ion content of bioceramics in simulated body environment is an effective approach to investigate the dissolution characteristics of materials [41]. Variations of calcium and phosphorus concentration as a function of time in the SBF solution under static and dynamic conditions were presented in Figs. 5 and 6(a). The changes in calcium ion concentration included the increase by the TCP dissolution and the decrease by the apatite precipitation. The Ca2+ concentration of the testing solution increased with immersion time in all TCP groups. It implied that the TCP dissolution dominated the reaction in SBF before the Ca ionic concentration reached the apex during the 12 days of immersion. It was remarkable that the Ca and [P] elements released from α-TCP dominated throughout the test, and β-TCP displayed the lowest degradation rate. The order of dissolution rate for the five samples was as follows: α-TCP N α8/β2-TCP N α5/β5-TCP N α2/β8-TCP N β-TCP, indicating that higher α-TCP phase in the α/β-TCP contributed to increased degradation. In fact, bone is surrounded in tissue fluid, which accounts for about 15% of adult body weight, and it is constantly circulating in the body to maintain homeostasis. Thus, the implanted bone substitutes always remain in a dynamic equilibrium with the body fluids. In order to simulate tissue fluid dynamics and investigate the dynamic degradation of the porous TCP bioceramics, a peristaltic pump circulation device was used (Fig. 7). As shown in Fig. 5b, the curve of Ca2+ concentration in dynamic environment was similar to that in static SBF. However, different from static condition, within the first day Ca release increased abruptly, and slowed down after 7 days under dynamic environment. Similar phenomena were found in [P] concentration under both static and dynamic conditions as shown in Fig. 6(a). Besides, the evolution of the pH values in SBF vs. soaking time under both conditions of assay was also shown in Fig. 6(b). The pH values in SBF solution exhibited a slight decrease from about 7.38 to 7.21–7.28 until 12 days due to the partial depletion of OH− in solution for the formation of new apatite deposition. The degradation rate for the five TCPs also decreased in the order α-TCP N α8/β2-TCP N α5/β5-TCP N α2/β8-TCP N β-TCP, and the level of Ca concentration in α8/β2-TCP sample was about 1.11 and 1.33 times than that of α5/β5-TCP and α2/β8-TCP groups, respectively. The dissolution rate in dynamic condition was higher than that under static environment, consistent with previous studies suggesting that a dynamic environment significantly facilitated the degradation of biomaterials by constantly updating the ceramic surface [42]. It is well-known that the dissolution rate of a biomaterial depends on multiple aspects including microstructure, porosity and crystal phases. From the above discussion, the three porous α/β-TCP ceramics
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015
1011
Fig. 3. SEM images of the porous TCP hybrid bioceramics with different magnifications (×40; ×5000): (a) α-TCP, (b) β-TCP, (c) α8/β2-TCP, (d) α5/β5-TCP, (e) α2/β8-TCP.
had similar microstructure and porosity, therefore, it was suggested that the different constitution proportions of α and β TCP phases in materials could mainly contribute to the dissolution rates of α/β-TCP. The resorption of calcium phosphate biomaterials is proved to be beneficial to bone formation and free Ca2+ from calcium phosphate is considered as the origin of bioactivity. Some earlier studies, at the same time, conled to drastic alteration of firm that excessive dissolved Ca2+ and PO3− 4 the microenvironment combined with the deteriorated mechanical properties of calcium phosphate-based materials, disruption in the activity of host cells and creating adverse effects on adjacent tissues [43, 44]. Our porous α/β-TCP showed a steerable release of Ca2+ contents through altering the ratio of α and β TCP phases in the dual α/β-TCP bioceramics for optimizing degradation and osteoconductivity.
porous α5/β5-TCP bioceramic specimens after 12 days of immersion displayed a typical fine apatite microstructure under SEM, which was similar in composition and structure to bone apatite. It was observed that the crystals were poorly crystalline, consisting of plate-like crystals.
3.4. Bone-like apatite formation Since one of the significant characteristics of bioactive materials is their ability to bond with living bone through the formation of a bonelike apatite layer on their surfaces [45], the apatite formation on the porous dual α/β-TCP bioceramics after immersion in SBF under both static and dynamic conditions was assessed. As shown in Fig. 8, the
Fig. 4. Compressive strength of the porous α-TCP, β-TCP, and α/β-TCP biphasic bioceramics.
Fig. 5. The calcium ion concentrations degraded from various TCP bioceramics at different soaking time in static (a) and dynamic (b) SBF.
1012
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015
Fig. 6. Variation of phosphorous content (a) (after subtracting the initial [P] concentration of SBF) and pH value (b) with soaking time in SBF for TCP bioceramics under static and dynamic conditions.
Fig. 7. Schematic diagram of the dynamic degradation for the porous α/β-TCP bioceramics under dynamic SBF.
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015
1013
Fig. 8. SEM images of the porous α5/β5-TCP bioceramics acquired after soaking in static (a–b) and dynamic SBF (c–d) for 12 days with different magnifications.
From the high-resolution images of Fig 7b and d, the plates coalesced to form large apatite clusters with flower-like structure, and particles became interconnected on the surface of macropore in scaffolds. However, a continuous release of Ca and P could slow the reactivity of materials. The formation of the bone-like apatite layer may involve cation substitutions at the Ca2 + sites, and/or the substitution at PO34 − sites by ions, with the concomitant creation of calcium vacancies to satHPO2− 4 isfy the condition of electrical charge neutrality [46]. The SEM analysis revealed that the dissolution and precipitation process resulted in calcium phosphate globule formation, and the formation of a new apatite layer after immersion of the specimens in SBF confirmed that the porous α5/β5-TCP hybrid bioceramics had a good in vitro bioactivity, which has been thought to be one of the necessary conditions for biomedical applications. 3.5. Dissolution kinetics of the porous α/β-TCP biphasic bioceramics Next, another goal of the work is to present and calculate the dissolution kinetics of the porous dual α/β-TCP bioceramics to understand their degradation attributes. The dissolution kinetics of calcium phosphate can be described by a single or a mixture of the three following rate laws: linear, parabolic, and exponential. These three types of kinetics are explained by the following rate-determining mechanisms, respectively: (a) diffusion or adsorption, (b) surface spiral dissolution, and (c) surface nucleation [47]. In the present study, the dissolution of the porous biphasic α/β-TCP bioceramic is assumed to be diffusion controlled. On the bases of the dissolution results, the classic Avrami equation is put forward to describe the kinetics of the porous α/β-TCP bioceramics, and the kinetic model is expressed by: f ¼ 1 exp: −kt
n
where f is the dissolution rate of TCP bioceramics at different immersion time t, k is rate constant, and n represents the Avrami exponent which is a coefficient related to the material property and shape. This can be rewritten as: − ln ð1 f Þ ¼ kt
n
Furthermore, there are three types of dissolution kinetics model according to the value of “n” in the equation: (i) n b 1, the starting dissolution rate is infinite and the dissolution rate decreases with time, (ii) n = 1, the starting dissolution rate is finite, and (iii) n N 1, the starting dissolution rate is near zero, respectively. Obviously, the research model of the porous α/β-TCP hybrid bioceramics belonged to type (ii) with finite stating dissolution rate. The kinetic equation became y = −ln (1 − f ) = kt, and the logarithm of (1 − f ) is a linear function of time (t). In the current experimental conditions, the porous monophasic and biphasic TCP bioceramics gradually degraded in SBF until the concentration of calcium ion reached Cmax (the saturation value). Cmax values for α-TCP, α8/β2-TCP, α5/β5-TCP, α2/β8-TCP and βTCP were about 29.5, 28.3, 27.6, 26.7 and 26.1 mg/L after immersion in SBF for 30 days, respectively. Hence, f was calculated by CTCP/Cmax, in which CTCP represented the current concentration of calcium ion released from various porous TCP bioceramics. The resultant −ln (1 − f ) was plotted as a function of t as shown in Fig. 9, and the y value showed a good linear relation with immersion time (t) (with R2 value N 0.987), whose y-intercept approximated to zero, and slope represented k value (rate constant for five TCP bioceramics). The dissolution profile of the porous α/β-TCP was, thereby, well expressed by the equation −ln (1 − f ) = kt, and the rate constant (k) was calculated from the slope of curve displayed in Table S2. It could be found that the rate constant for the porous α/β-TCP increased with the contents of α-TCP phase. Namely, increased α-TCP levels
1014
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015
Acknowledgments This work was supported by the Open Fund of Sichuan Province Key Laboratory of Nonmetal Composite and Functional Materials (No. 11zxfk11).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.11.040.
References
Fig. 9. Linearization of the various porous TCP bioceramics dissolution under static (a) and dynamic (b) SBF using the Avrami equation y = −ln (1 − f) = kt.
promoted the degradation of α/β-TCP regardless of under static and dynamic environments, further confirming that the dissolution behavior of the porous biphasic complex could be steered depending on the components of α and β phases. Through fitting of the dissolution rate with the Avrami equation, the desired Ca2+ content, which is needed or sufficient for bone formation in the human body released from biphasic α/β-TCP at different time points, could be predicted, providing a help to design the porous α/β-TCP bioceramics for bone tissue engineering. 4. Conclusions Overall, the ACP-derived porous TCP hybrid bioceramics displayed a homogeneous microstructural character of interconnected macropore structure and the co-existence of α-TCP and β-TCP phases in each α/ β-TCP. Bioactivity and dissolution behaviors in SBF were conducted by immersing the scaffold specimens for 12 days. The porous α/β-TCP scaffolds showed good in vitro bioactivity due to the formation of bone-like apatite phase on the surface. The dissolution results showed that dynamic environment enhanced the dissolution of α/β-TCP than that under static condition. The porous biphasic α/β-TCP bioceramics with adjustable degradation rate were obtained via tuning the proportion of α- and β-TCP content, and the dissolution rate of porous TCP biomaterials also could be well-expressed and fitted using the Avrami formula (−ln (1 − f ) = kt) for an ionic diffusion model. Due to excellent bioactivity and controlled degradability, therefore, the porous biphasic α/β-TCP bioceramics could offer promising potential for the repair and replacement of bone.
[1] R. Langer, D.A. Tirrell, Designing materials for biology and medicine, Nature 428 (2004) 487–492. [2] A.K. Gaharwar, S.M. Mihaila, A. Swami, A. Patel, S. Sant, R.L. Reis, A.P. Marques, M.E. Gomes, A. Khademhosseini, Bioactive silicate nanoplatelets for osteogenic differentiation of human mesenchymal stem cells, Adv. Mater. 25 (2013) 3329–3336. [3] R.R. Betz, Limitations of autograft and allograft: new synthetic solutions, Orthopedics 25 (2002) s561–s570. [4] B.M. Holzapfel, J.C. Reichert, J.-T. Schantz, U. Gbureck, L. Rackwitz, U. Noeth, F. Jakob, M. Rudert, J. Groll, D.W. Hutmacher, How smart do biomaterials need to be? A translational science and clinical point of view, Adv. Drug Deliv. Rev. 65 (2013) 581–603. [5] S. Higashi, T. Yamamuro, T. Nakamura, Y. Ikada, S.-H. Hyon, K. Jamshidi, Polymerhydroxyapatite composites for biodegradable bone fillers, Biomaterials 7 (1986) 183–187. [6] E. Willbold, X. Gu, D. Albert, K. Kalla, K. Bobe, M. Brauneis, C. Janning, J. Nellesen, W. Czayka, W. Tillmann, Effect of the addition of low rare earth elements (lanthanum, neodymium, cerium) on the biodegradation and biocompatibility of magnesium, Acta Biomater. 11 (2015) 554–562. [7] W. Suchanek, M. Yoshimura, Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants, J. Mater. Res. 13 (1998) 94–117. [8] U. Ripamonti, L.C. Roden, L.F. Renton, Osteoinductive hydroxyapatite-coated titanium implants, Biomaterials 33 (2012) 3813–3823. [9] B. Liu, D.X. Lun, Current application of β-tricalcium phosphate composites in orthopaedics, Orthop. Surg. 4 (2012) 139–144. [10] H.-S. Ryu, K.S. Hong, J.-K. Lee, D.J. Kim, J.H. Lee, B.-S. Chang, D.-h. Lee, C.-K. Lee, S.-S. Chung, Magnesia-doped HA/β-TCP ceramics and evaluation of their biocompatibility, Biomaterials 25 (2004) 393–401. [11] M. Yashima, A. Sakai, High-temperature neutron powder diffraction study of the structural phase transition between α and α′ phases in tricalcium phosphate Ca3(PO4)2, Chem. Phys. Lett. 372 (2003) 779–783. [12] X. Wei, O. Ugurlu, A. Ankit, H.Y. Acar, M. Akinc, Dissolution behavior of Si, Zncodoped tricalcium phosphates, Mater. Sci. Eng. C Mater. 29 (2009) 126–135. [13] N. Matsumoto, K. Yoshida, K. Hashimoto, Y. Toda, Thermal stability of β-tricalcium phosphate doped with monovalent metal ions, Mater. Res. Bull. 44 (2009) 1889–1894. [14] I. Martinez, P. Velasquez, L. Meseguer-Olmo, A. Bernabeu-Esclapez, P. De Aza, Preparation and characterization of novel bioactive α-tricalcium phosphate doped with dicalcium silicate ceramics, Mater. Sci. Eng. C Mater. 32 (2012) 878–886. [15] H. Yuan, J. De Bruijn, Y. Li, J. Feng, Z. Yang, K. De Groot, X. Zhang, Bone formation induced by calcium phosphate ceramics in soft tissue of dogs: a comparative study between porous α-TCP and β-TCP, J. Mater. Sci. Mater. Med. 12 (2001) 7–13. [16] J. Lu, M. Descamps, J. Dejou, G. Koubi, P. Hardouin, J. Lemaitre, J.-P. Proust, The biodegradation mechanism of calcium phosphate biomaterials in bone, J. Biomed. Mater. Res. 63 (2002) 408–412. [17] N. Kondo, A. Ogose, K. Tokunaga, H. Umezu, K. Arai, N. Kudo, M. Hoshino, H. Inoue, H. Irie, K. Kuroda, Osteoinduction with highly purified β-tricalcium phosphate in dog dorsal muscles and the proliferation of osteoclasts before heterotopic bone formation, Biomaterials 27 (2006) 4419–4427. [18] A. Ogose, N. Kondo, H. Umezu, T. Hotta, H. Kawashima, K. Tokunaga, T. Ito, N. Kudo, M. Hoshino, W. Gu, Histological assessment in grafts of highly purified betatricalcium phosphate (OSferion®) in human bones, Biomaterials 27 (2006) 1542–1549. [19] J. Huang, E. Ten, G. Liu, M. Finzen, W. Yu, J.S. Lee, E. Saiz, A.P. Tomsia, Biocomposites of pHEMA with HA/β-TCP (60/40) for bone tissue engineering: swelling, hydrolytic degradation, and in vitro behavior, Polymer 54 (2013) 1197–1207. [20] X. Xiao, R. Liu, Q. Huang, X. Ding, Preparation and characterization of hydroxyapatite/ polycaprolactone–chitosan composites, J. Mater. Sci. Mater. Med. 20 (2009) 2375–2383. [21] N. Sultana, T.H. Khan, In vitro degradation of PHBV scaffolds and nHA/PHBV composite scaffolds containing hydroxyapatite nanoparticles for bone tissue engineering, J. Nanomater. 2012 (2012) 1. [22] L. Mao, G. Yuan, J. Niu, Y. Zong, W. Ding, In vitro degradation behavior and biocompatibility of Mg–Nd–Zn–Zr alloy by hydrofluoric acid treatment, Mater. Sci. Eng. C 33 (2013) 242–250. [23] K. Hagihara, S. Shakudo, K. Fujii, T. Nakano, Degradation behavior of Ca–Mg–Zn intermetallic compounds for use as biodegradable implant materials, Mater. Sci. Eng. C 44 (2014) 285–292.
L. Xie et al. / Materials Science and Engineering C 59 (2016) 1007–1015 [24] M. Houmard, Q. Fu, M. Genet, E. Saiz, A.P. Tomsia, On the structural, mechanical, and biodegradation properties of HA/β-TCP robocast scaffolds, J. Biomed. Mater. Res. B. Appl. Biomater. 101 (2013) 1233–1242. [25] C. Shuai, J. Zhuang, H. Hu, S. Peng, D. Liu, J. Liu, In vitro bioactivity and degradability of β-tricalcium phosphate porous scaffold fabricated via selective laser sintering, Biotechnol. Appl. Biochem. 60 (2013) 266–273. [26] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 27 (2006) 2907–2915. [27] Y. Yang, Y. Zhao, G. Tang, H. Li, X. Yuan, Y. Fan, In vitro degradation of porous poly (Llactide-co-glycolide)/β-tricalcium phosphate (PLGA/β-TCP) scaffolds under dynamic and static conditions, Polym. Degrad. Stab. 93 (2008) 1838–1845. [28] J.-p. Seo, N. Tsuzuki, S. Haneda, K. Yamada, H. Furuoka, Y. Tabata, N. Sasaki, Proliferation of equine bone marrow-derived mesenchymal stem cells in gelatin/βtricalcium phosphate sponges, Res. Vet. Sci. 93 (2012) 1481–1486. [29] S. Kobayashi, S. Yamadi, Strain rate dependency of mechanical properties of TCP/ PLLA composites after immersion in simulated body environments, Compos. Sci. Technol. 70 (2010) 1820–1825. [30] S.-H. Kwon, Y.-K. Jun, S.-H. Hong, H.-E. Kim, Synthesis and dissolution behavior of βTCP and HA/β-TCP composite powders, J. Eur. Ceram. Soc. 23 (2003) 1039–1045. [31] Y. Li, W. Weng, K.C. Tam, Novel highly biodegradable biphasic tricalcium phosphates composed of α-tricalcium phosphate and β-tricalcium phosphate, Acta Biomater. 3 (2007) 251–254. [32] M. Kamitakahara, C. Ohtsuki, T. Miyazaki, Review paper: behavior of ceramic biomaterials derived from tricalcium phosphate in physiological condition, J. Biomater. Appl. 23 (2008) 197–212. [33] M. Kamitakahara, C. Ohtsuki, M. Oishi, S. Ogata, T. Miyazaki, M. Tanihara, Preparation of porous biphasic tricalcium phosphate and its in vivo behavior, Key Eng. Mater. 281-284 (2005). [34] R. LeGeros, S. Lin, R. Rohanizadeh, D. Mijares, J. LeGeros, Biphasic calcium phosphate bioceramics: preparation, properties and applications, J. Mater. Sci. Mater. Med. 14 (2003) 201–209. [35] K. Sariibrahimoglu, J.G. Wolke, S.C. Leeuwenburgh, L. Yubao, J.A. Jansen, Injectable biphasic calcium phosphate cements as a potential bone substitute, J. Biomed. Mater. Res. B. Appl. Biomater. 102 (2014) 415–422.
1015
[36] J. Jansen, J. Vehof, P. Ruhe, H. Kroeze-Deutman, Y. Kuboki, H. Takita, E. Hedberg, A. Mikos, Growth factor-loaded scaffolds for bone engineering, J. Control. Release 101 (2005) 127–136. [37] G.B. Bishop, T.A. Einhorn, Current and future clinical applications of bone morphogenetic proteins in orthopaedic trauma surgery, Int. Orthop. 31 (2007) 721–727. [38] J. Zeltinger, J.K. Sherwood, D.A. Graham, R. Müeller, L.G. Griffith, Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition, Tissue Eng. 7 (2001) 557–572. [39] O. Gauthier, J.-M. Bouler, E. Aguado, P. Pilet, G. Daculsi, Macroporous biphasic calcium phosphate ceramics: influence of macropore diameter and macroporosity percentage on bone ingrowth, Biomaterials 19 (1998) 133–139. [40] H. Kiba, N. Kuboyama, R. Uchida, T. Ishizaki, N. Nishiyama, Bone ingrowth into the parallel cylindrical tubes with different sizes of porous hydroxyapatite implanted into the rabbits, J. Hard Tissue Biol. 21 (2012) 307–314. [41] Q. Wang, Q. Wang, J. Wang, X. Zhang, X. Yu, C. Wan, Degradation kinetics of calcium polyphosphate bioceramic: an experimental and theoretical study, Mater. Res. 12 (2009) 495–501. [42] C. Agrawal, J. McKinney, D. Lanctot, K. Athanasiou, Effects of fluid flow on the in vitro degradation kinetics of biodegradable scaffolds for tissue engineering, Biomaterials 21 (2000) 2443–2452. [43] T. Suzuki, T. Yamamoto, M. Toriyama, K. Nishizawa, Y. Yokogawa, M.R. Mucalo, Y. Kawamoto, F. Nagata, T. Kameyama, Surface instability of calcium phosphate ceramics in tissue culture medium and the effect on adhesion and growth of anchorage-dependent animal cells, J. Biomed. Mater. Res. 34 (1997) 507–517. [44] S. Yamada, D. Heymann, J.-M. Bouler, G. Daculsi, Osteoclastic resorption of calcium phosphate ceramics with different hydroxyapatite/β-tricalcium phosphate ratios, Biomaterials 18 (1997) 1037–1041. [45] T. Kokubo, H.M. Kim, M. Kawashita, Novel bioactive materials with different mechanical properties, Biomaterials 24 (2003) 2161–2175. [46] Y.F. Chou, W.A. Chiou, Y. Xu, J.C. Dunn, B.M. Wu, The effect of pH on the structural evolution of accelerated biomimetic apatite, Biomaterials 25 (2004) 5323–5331. [47] R. Tang, W. Wu, M. Haas, G.H. Kinetics of dissolution of β-tricalcium phosphate, Langmuir 17 (2001) 3480–3485.