Aug 9, 2007 - 1Mechanical Engineering Department, Istanbul Technical University, Taksim, ... For samples with 1.6 Ca/P ratios, the HA phase was domi-.
Increased osteoblast adhesion on nanoparticulate calcium phosphates with higher Ca/P ratios Celaletdin Ergun,1 Huinan Liu,2 Thomas J. Webster,2 Ercan Olcay,3 S˛afak Yılmaz,1 Filiz C. Sahin4 1 _ Mechanical Engineering Department, Istanbul Technical University, Taksim, Istanbul, Turkey 2 Divisions of Engineering and Orthopaedics, Brown University, Providence, Rhode Island 02912 3 _ Vakıf Gruba Educational Hospital, Department of Orthopedic and Traumatalogy, Vatan Caddesi, Istanbul, Turkey 4 _ Istanbul Technical University, Prof. Dr. Adnan Tekin High Technical Ceramic and Composite Research, Maslak, Istanbul, Turkey Received 10 November 2006; revised 15 February 2007; accepted 5 June 2007 Published online 9 August 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31555 Abstract: The biological properties of calcium phosphatederived materials are strongly influenced by changes in Ca/P stoichiometry and grain size, which have not yet been fully elucidated to date. For this reason, the objective of this in vitro study was to understand osteoblast (bone forming cells) adhesion on nanoparticulate calcium phosphates of various Ca/ P ratios. A group of calcium phosphates with Ca/P ratios between 0.5 and 2.5 were obtained by adjusting the Ca/P stoichiometry of the initial reactants necessary for calcium phosphate precipitation. For samples with 0.5 and 0.75 Ca/P ratios, tricalcium phosphate (TCP) and Ca2P2O7 phases were observed. In contrast, for samples with 1.0 and 1.33 Ca/P ratios, the only stable phase was TCP. For samples with 1.5 Ca/P ratios, the TCP phase was dominant, however, small amounts of the hydroxyapatite (HA) phase started to appear. For samples with 1.6 Ca/P ratios, the HA phase was domi-
nant. Last, for samples with 2.0 and 2.5 Ca/P ratios, the CaO phase started to appear in the HA phase, which was the dominant phase. Moreover, the average nanometer grain size, porosity (%), and the average pore size decreased in general with increasing Ca/P ratios. Most importantly, results demonstrated increased osteoblast adhesion on calcium phosphates with higher Ca/P ratios (up to 2.5). In this manner, this study provided evidence that Ca/P ratios should be maximized (up to 2.5) in nanoparticulate calcium phosphate formulations to increase osteoblast adhesion, a necessary step for subsequent osteoblast functions such as new bone deposition. Ó 2007 Wiley Periodicals, Inc. J Biomed Mater Res 85A: 236–241, 2008
INTRODUCTION
will depend to a great extent on their ratios of calcium (Ca) to phosphorous (P). Moreover, the Ca/P ratios of calcium phosphates in bulk or in coatings vary according to which of the following phases are present: aand b-tricalcium phosphate (TCP), tetracalcium phosphate, octacalcium phosphate, and hydroxyapatite (HA or Ca10(PO4)6(OH)2). In addition, less crystalline phases of calcium phosphates (such as TCP) degrade much faster than the crystalline phase HA.5 Among these phases, pure crystalline HA is known to be the most stable and strongest phase.6 Although it has been documented that the bone apposition is significantly improved at the surface of an HA coated compared to an uncoated metallic implant (thus, providing a stronger bone–implant interface7), other types of more degradable calcium phosphates may be desirable in orthopedic applications such as drug delivery, bone grafting, and as biodegradable bone cement materials. Thus, to match the rate of new bone formation, a biphasic HA/TCP ceramic may be more advantageous.8 In addition to
Calcium phosphates have been used as bulk implants or as coatings on orthopedic and dental implants to achieve fast chemical bonding between bone and an implant.1,2 Calcium phosphate materials may degrade in extracellular fluids due to an acidic wound healing response and/or by cellular activity within compartments of low pH.3 As a result, concerns have been raised about the release of calcium phosphate debris after implantation, which will influence calcium phosphate coating stability and strength at the bone–implant interface.4 Importantly, the longterm stability of calcium phosphate derived materials Correspondence to: T. J. Webster; e-mail: Thomas_Webster@ Brown.edu Contract grant sponsors: Istanbul Technical UniversityBAP program and the Scientific and Technological Research Council of Turkey-BAYG program ' 2007 Wiley Periodicals, Inc.
Key words: osteoblasts; calcium phosphates; bioceramics; biomaterials; orthopedic implants; nanoparticles
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TABLE I The Abbreviation, Composition, and Characterization of the Various Calcium Phosphate Samples Used in this Study Composition (at %) Sample Abbreviation
Ca
P
O
Grain Size (nm)
% Porosity
Pore Size (nm)
CP05 CP075 CP10 CP13 CP15 CP16 CP20 CP25
45.15 28.12 26.24 32.46 27.88 36.34 36.01 48.77
24.96 20.31 18.8 22.07 19.34 22.8 14.96 18.59
29.89 51.57 54.95 45.47 52.78 40.86 49.04 32.64
1370 1300 1830 1437 819 290 575 262
14.8 7.2 6.2 3.3 3.1 2.9 3.3 4.9
1010 630 580 410 380 260 220 190
CP, calcium/phosphate and numbers correspond to the Ca/P ratio in each specific sample.
intentionally designing calcium phosphate materials to be more biodegradable or more stable, there are several unintentional cases that may lead to a lack of purity in the produced HA phase. For example, many coating processes lead to bulk or localized Ca/P ratios that can deviate from the standard HA stoichiometric value of 1.67. Calcium oxide (CaO) can be induced from either thermal decomposition9 or from intentional additions for improving thermal stability.10 TCP is another common product of thermal decomposition that may occur during HA coating processes. Clearly, whether intentional or not, variations in Ca/ P ratios could produce inconsistent biological responses in vitro or in vivo. Therefore, to choose appropriate calcium phosphates for specific medical applications, it is crucial to characterize the biological properties of calcium phosphates with various Ca/P ratios. For this reason, the objective of this in vitro study was to elucidate the relationship between calcium phosphates with various Ca/P ratios and osteoblast adhesion. Because of recent promising results of greater osteoblast adhesion on nanoparticulate calcium phosphates (such as HA11,14–16), this study varied Ca/P ratios in calcium phosphate-based nanoparticles. Wet chemistry precipitation methods were used to produce synthetic calcium phosphates of various Ca/P ratios (between 0.5 and 2.5) by adjusting the concentration of the initial reactants. Characterization of various calcium phosphates was completed by X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). Moreover, osteoblast adhesion was determined in vitro to ascertain their cytocompatibility properties. Adhesion of anchorage-dependent cells (such as osteoblasts) is a prerequisite for subsequent cell functions, such as the deposition of calcium-containing mineral. MATERIALS AND METHODS Synthesis of calcium phosphates HA was synthesized by established wet chemistry precipitation methods.11 First, stock solutions were prepared in
different bottles by dissolving ammonium phosphate and calcium nitrate in distilled water. The two solutions were mixed at pH levels of 11–12. The mixing ratio was adjusted according to the desired Ca/P ratios in the precipitated calcium phosphates. Next, the mixture was stirred for several days. The slurry was filtered using a filter paper and dried at 608C for several days. The precipitated calcium phosphates were crushed with a mortar and pestle, and sieved through a 200-mesh screen to obtain powders, which have particle sizes less than 75 lm in diameter. This powder was then dry pressed into pellets and fired at 11008C for 2 h. The composition and the description of the samples of interest to this study are summarized in Table I.
Materials characterization X-ray diffraction spectra of the calcium phosphates with various Ca/P ratios were collected using a Rigaku Rint Dmax 100 diffractometer with Co Ka radiation. XRD was run with 2u angles between 208 and 508 with a 0.028/min scan speed at 40 kV and 30 mA. Scanning electron microscope (SEM) examinations and elemental analysis were performed with a Philips XL30 FEG SEM. Quantitative image analysis techniques were selected to evaluate the grain size, porosity, and pore size of all samples based on obtained SEM micrographs. The linear intercept method12,13 was used to determine the average grain size of various calcium phosphates of interest. The areal analysis technique was used to determine the porosity and the average pore size of various calcium phosphate samples.
Cytocompatibility tests Cell culture Human osteoblasts (bone-forming cells; CRL-11372 American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1% penicillin/streptomycin (P/S; Hyclone) under standard cellculture conditions, that is, a sterile, 378C, humidified, 5% CO2/95% air environment. Cells at population numbers 6–9 were used in the experiments. Journal of Biomedical Materials Research Part A
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RESULTS Materials characterization X-ray diffraction patterns of the precipitated calcium phosphates of various Ca/P ratios are presented in Figure 1. X-ray diffraction peaks in all of the precipitated samples matched JSPD standard peaks of calcium phosphate hydrate (Ca3(PO4)2 3 H2O). The background showed the existence of possible amorphous calcium phosphate phases. These phases started to crystallize and transformed into related calcium phosphate phases depending on their Ca/P ratios upon sintering at 11008C. X-ray diffraction patterns of the calcium phosphates of various Ca/P ratios after sintering at 11008C are presented in Figure 2. Results demonstrated that CP05 and CP075 samples possessed two phases: b-TCP and Ca2P2O7. For CP10 and CP13, the only stable phase was b-TCP. For CP15, slight HA formation was observed, while for CP16, the only stable phase was HA. For CP20, the formation of CaO was detected, and the
Figure 1. XRD patterns of the following precipitated calcium phosphates: (a) CP05; (b) CP075; (c) CP10; (d) CP13; (e) CP15; (f) CP16; (g) CP20; and (h) CP25. (!: calcium phosphate hydrate). Y-axis ¼ arbitrary units.
Osteoblast adhesion All sterilized calcium phosphate pellets listed in Table I were placed in 12-well tissue culture plates (Corning) and rinsed thrice with sterilized phosphate buffered saline (PBS; a solution containing 8 g NaCl, 0.2 g KCl, 1.5 g Na2HPO4, and 0.2 g KH2PO4 in 1000 mL deionized water adjusted to a pH of 7.4; all chemicals from Sigma). Osteoblasts were seeded at a concentration of 2500 cells/ cm2 onto the samples of interest in DMEM supplemented with 10% FBS and 1% P/S and then incubated under standard cell-culture conditions for 4 h. After that time period, nonadherent cells were removed by rinsing with PBS, and adherent cells were then fixed with formaldehyde (Fisher Scientific) and stained with Hoechst 33258 dye (Sigma); the cell nuclei were, thus, visualized and counted under a fluorescence microscope (Leica, excitation wavelength 365 nm and emission wavelength 400 nm). Cell counts were expressed as the average number of cells on eight random fields per sample. All experiments were run in triplicate at least three times, and cell adhesion was evaluated based on the mean number of adherent cells. Numerical data were analyzed using standard analysis of variance (ANOVA) techniques and Student t test; statistical significance was considered at p < 0.05. Journal of Biomedical Materials Research Part A
Figure 2. XRD patterns of the following calcium phosphates sintered at 11008C: (a) CP05; (b) CP075; (c) CP10; (d) CP13; (e) CP15; (f) CP16; (g) CP20; and (h) CP25. (^: HA; l: CaO; !: b-TCP (b-Ca3(PO4)6); : Ca2P2O7). Y-axis ¼ arbitrary units.
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Figure 3. SEM micrographs of the following calcium phosphates sintered at 11008C: (a) CP05; (b) CP075; (c) CP10; (d) CP13; (e) CP15; (f) CP16; (g) CP20; and (h) CP25. Scale bars: 5 lm.
amount of this phase increased in CP25, although HA was the dominant phase in both CP20 and CP25. Thus, as the Ca/P ratio increased in the samples, the phases present followed this progression: b-TCP þ Ca2P2O7 ? b-TCP ? b-TCP þ HA ? HA ? HA þ CaO.
The microstructures of the samples are shown in Figure 3. Quantitative analysis of these SEM images provided evidence that the average grain sizes, porosity, and the average pore sizes decreased in general while Ca/P ratios increased (Table I). For example, Journal of Biomedical Materials Research Part A
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Figure 4. Increased osteoblast adhesion on nanoparticulate calcium phosphates with higher Ca/P ratios. Values are mean 6 SEM; n ¼ 3; *p < 0.05 (compared to CP05 through CP20); **p < 0.05 (compared to CP05 through CP15); ***p < 0.05 (compared to CP05 and CP075); and #p < 0.05 (compared to CP075).
CP05 had grain sizes of 1370 nm, porosity of 14.8%, and pore sizes of 1010 nm while CP25 had grain sizes of 575 nm, porosity of 3.3%, and pore sizes of 220 nm. Osteoblast adhesion Results of this study showed that osteoblast adhesion increased on the calcium phosphates with higher Ca/P ratios (Fig. 4). Generally, osteoblast adhesion on calcium phosphates can be divided into four groups. That is, the first group included CP05 and CP075; the second group included CP10, CP13, and CP15; the third group included CP16 and CP20; and the fourth group included CP25. Osteoblast adhesion increased significantly from the first group to the fourth group while no significant differences in osteoblast adhesion were detected within each group. Specifically, osteoblast adhesion on CP25 (the fourth group) was 3.5 times higher than CP05 and CP075 (the first group), 2.3 times higher than CP10, CP13, and CP15 (the second group), and 1.75 times higher than CP16 and CP20 (the third group). Osteoblast adhesion on CP16 and CP20 (the third group) was two times higher than CP05 and CP075 (the first group) and 1.3 times higher than CP10, CP13, and CP15 (the second group). However, no significant differences in osteoblast adhesion was observed between CP05 and CP075 (within the first group), among CP10, CP13, and CP15 (within the second group), or between CP16 and CP20 (within the third group). Overall, calcium phosphates with the highest Ca/P ratio (that is, CP25) increased osteoblast adhesion the most. DISCUSSION Results of this in vitro study provided evidence of increased osteoblast adhesion on nanoparticulate calcium phosphates with greater Ca/P ratios (up to 2.5). Journal of Biomedical Materials Research Part A
Such information is useful when designing calcium phosphate materials for orthopedic implant coatings, drug delivery applications, and degradable bone cements. For example, while HA is the most stable phase to create a bioactive coating on an orthopedic implant, the highest Ca/P ratio in HA formulations would promote the most osteoblast adhesion. Similarly, for ceramic drug-delivery applications, less crystalline materials are desirable so as to degrade and release embedded bone-building agents, thus, amorphous materials with the highest Ca/P ratios would promote osteoblast adhesion on those substrates. Clearly, there are chemistry changes in the nanoparticulate calcium phosphates of various Ca/P ratios formulated in this study. Since many proteins important for osteoblast adhesion (such as vitronectin) have calcium binding sites, surface chemistry changes of higher Ca/P ratios may influence initial protein interactions, which are important for mediating subsequent osteoblast adhesion. Such higher amounts of calcium on surfaces may also serve as mineral nucleation sites beneficial for long-term osseointegration events. However, additional material properties may also have influenced osteoblast adhesion on the substrates of higher Ca/P ratios, such as decreased grain size into the nanometer regime and decreased porosity and pore size, which may have altered surface energetics, area and degree of nanoscale surface roughness. Specifically, quantitative analysis of SEM micrographs revealed that for an increase in Ca/P ratio, smaller nanometer grain sizes accompanied by a decrease in porosity and pore size resulted. In this manner, increased osteoblast adhesion on calcium phosphates with smaller nanometer grain sizes confirms other reports in the literature, which have shown greater osteoblast adhesion, proliferation, alkaline phosphatase synthesis, and calcium deposition on pure HA with 67 compared to 167 nm grain sizes.14 Recent studies have furthered this in vitro data and demonstrated increased infiltration of rat calvaria bone into tantalum scaffolds coated with nanometer compared to conventional HA as early as 2 weeks.15 Such promoted osteoblast functions on smaller nanometer grain size HA was attributed to their unique surface properties (due to increased numbers of grain boundaries, surface defects, etc.) that increased the adsorption of proteins known to promote osteoblast adhesion (such as vitronectin).16 The same events may be happening here. Since it is well established that HA with greater nanoscale roughness (due to the decreased grain size) increases bone formation, topography may be an important material property promoting osteoblast adhesion on the presently studied substrates. Nonetheless, while our study provides design criteria to increase the performance of numerous types of
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orthopedic materials, further investigation is needed to determine an exact mechanism as to why the trends observed in this study are occurring, such as by examining altered initial protein interactions on calcium phosphates with higher Ca/P ratios. Also, it is important to keep in mind that although osteoblast adhesion is a necessary step for osteoblast long-term functions (such as calcium deposition), more studies are clearly needed to determine subsequent functions of osteoblasts on the substrates of interest to this study.
CONCLUSIONS Results of this in vitro study demonstrated increased osteoblast adhesion on nanoparticulate calcium phosphates with higher Ca/P ratios of up to 2.5. In this manner, this study provided evidence that Ca/P ratios should be maximized (up to 2.5) in nanoparticle calcium phosphate formulations to increase osteoblast adhesion, a necessary step for subsequent osteoblast functions such as bone deposition.
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Journal of Biomedical Materials Research Part A
