Tailoring of multilayered core-shell nanostructure for

Tailoring of multilayered core-shell nanostructure for multicomponent administration and controllable release of biologically active ions Zhongru Gou,1 Wenjian Weng,1 Piyi Du,1 Gaorong Han,1 Weiqi Yan2 1 Department of Materials Science and Engineering, Zhejiang University, Zheda Road 37, Hangzhou 310027, China 2 Orthopaedic Research Center, Second Affiliated Hospital of School of Medicine, Zhejiang University, Jiefang Road 88, Hangzhou 310006, China Received 12 April 2006; revised 23 February 2007; accepted 9 May 2007 Published online 26 September 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31582 Abstract: The biomaterials that can control the active ions delivery to enhance cell activity are regarded as promising bone regenerative materials. In this study, a new approach aiming to layer-by-layer (LbL) assemble the bioavailable zinc ions in the core-shell-like silica@octacalcium phosphate (OCP) nanosphere and to analyze its efficacy on improving controlled-release was reported. Firstly, a pHresponsive electrostatic interaction was used to adsorb zinc ions on silica nanospheres with different zinc concentration, which was followed by coating silica gel layer. Then the nanospheres were LbL assembled with zinc ions and silica gel alternately until the desired multilayered nanospheres were achieved. Finally, the porous OCP shells were capped onto the outside surface of the nanospheres

tailored by poly(aspartic acid) sodium molecules. The ion release tests in Tris buffers in vitro indicated that zinc release was controlled by pH and storage capacity, and silicon release was regulated by the OCP shell barrier. A temporal gradient within short times and sustained-dosage for a prolonged time toward the zinc and silicon ions could be obtained in this multilayer system. The results of this organized active ion assembly might open a promising future direction for effective delivery of trace elements in bone defect therapy. Ó 2007 Wiley Periodicals, Inc. J Biomed Mater Res 85A: 909–918, 2008

INTRODUCTION

highly dependent on the delivered silicon dose, and additionally, the role of zinc in bone mineralization is also associated with its dose ranges. A variety of recent studies indicate that a temporal overhigh silicon doses leaching from bioactive glasses (*200 ppm) result in medium alkalization, cell vacuolization and apoptosis, and/or inhibiting cell proliferation.17–22 It is generally accepted that cell proliferation and bone remodeling are retarded without adequate zinc dosage,2,3 whereas zinc can elicit counterproductive responses, such as bone resorption and turnover, rather than bone regeneration if used in excessive doses (>3 ppm).23,24 Conventional methods to fabricate bioactive glasses are mainly relied on high-temperature melting and sol-gel techniques, whereas the factors influencing the active ion release are complicated.25–29 Currently, much effort has been made to show that the individual trace element lattice substitution in calcium phosphate (CaP) bioceramics represents a breakthrough in enhancing osteoconduction of conventional bone substitutes.30,31 Nonetheless, this method is also inadequate to meet the needs for a

Trace elements have been attracting considerable interest in biomaterials due to their potential pharmaceutical effect and stimulatory gene expression activity.1–9 The beneficial effects of silicon are mainly associated with its high deposition in young bone tissues.6,7 Some silica-based bioactive glasses exhibit potential ability to activate gene expression,8,9 stimulate tissue regeneration,10 and even direct differentiation of stem cells to osteogenic cell lineages.11–13 Similarly, zinc can increase bone protein and DNA contents and enhance bone strength.14–16 However, the silicon activation effect on gene expression is Correspondence to: W. Weng; e-mail: [email protected] Contract grant sponsor: National 973 Projects, The Ministry of Science and Technology of P.R. China; contract grant number: 2002CB613302 Contract grant sponsor: National 863 Program, The Ministry of Science and Technology of P.R. China; contract grant number: 2003AA302220 ' 2007 Wiley Periodicals, Inc.

Key words: core-shell structure; trace element; layer-bylayer assembly; controlled release; bioactive materials

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better control of multicomponent administration, so that the release kinetics of biologically active ions cannot be controlled in an expected manner to mediate cell activity and accelerate implant bioabsorption. Inspiration comes from the layer-by-layer (LbL) assembly technique and stimuli-responsive controlled-release strategy that can achieve a favorable component administration and controllable release pattern in response to specific stimuli.32,33 Herein, we attempt to develop a core-shell Silica@octacalcium phosphate (OCP) multilayer system for a better controlled-release of biologically active ions (Scheme 1). Firstly, silica gel is chosen as a reservoir because it has not only been well demonstrated a biocompatible, 100% biodegradable matrix as versatile carriers of controlled functional genes and drugs delivery,34,35 but also has negatively charged surface with negative silanol groups to reversibly condense metal ions with respect to pH in the solution.36,37 Secondly, the design on LbL assembly is an effective approach to create well-defined architecture for a better active ion administration. Thirdly, the porous OCP shells are capped on the external surface of the multilayer nanospheres to avoid silicon release quickly within a short time stage.

MATERIALS AND METHODS Reagents and materials Zinc nitrate (99.5 wt %), zinc chloride (>99 wt %), tetraethylorthosilicate (TEOS, 98%), sodium phosphate (Na3PO4, 99.5 wt %), sodium silicate (Na2SiO3
H2O, 20.3 wt % SiO2), ammonia (NH4OH, 28 wt %), and tris(hydroxyllmethyl) aminomethane (Tris) were used as received from Shanghai Chemical Reagent (China). Poly(aspartic acid) sodium (PAAS)(average Mw 3.0 kDa, 30 wt % in water) was obtained from Taihe Water-Treatment Reagent (China). The CO2-excluded deionized water was used throughout the experiment.

Preparation of silica nanospheres The silica gel nanospheres (SGNs) were prepared with TEOS silica precursor and NH4OH in ethanol/water medium with some modification of the well-known Stober process.38 In a typical synthesis bath with anhydrous ethanol (17.80 mL) under vigorous stirring, a solution of NH4OH (0.72 mL) mixed with deionized water (0.58 mL) and Na2HPO4 (20 mM, 45 lL) was dissolved into this solution. Then 0.90 mL of TEOS was added quickly. Stirring was continued at room temperature for 6 h. The SGNs were then isolated from the excess nonaggregated silica sol by three cycles of centrifugation, followed by ultrasonically washed in deionized water. Journal of Biomedical Materials Research Part A

LbL assembly of Silica@Zn-x nanospheres The initial effort was focused on the assembly of zinc ions on the SGNs by modification of zinc concentration and pH in the 0.5 mmol/L KCl background electrolyte solution. The adsorption of zinc ions on SGNs (200 mg) in Zn(NO3)2 solutions (50 mL, pH ¼ 8.4, 7.8, or 7.4) with an initial zinc concentration (x) of 80, 400, or 2000 lmol/L were performed at 378C under stirring for equilibrium achievement. The pH was restored to the constant value by addition of the required amount of ammonia solution. Subsequently, the nanoparticles (denoted as Silica@Zn-x, x ¼ 80, 400, or 2000) were dispersed into 20 mL of freshly prepared sodium silicate aqueous solutions (0.54 wt %) with a pH of 8.4, 7.8, or 7.4 described as earlier and coated with a thin wall of silica gel film under magnetic stirring for 24 h. Thereafter, the resulting nanospheres (denoted as Silica@Zn-x@Silica) were assembled with zinc ions and silica gel alternately until the desired multilayer nanostructures were achieved.

Encapsulation of Silica@Zn-x@Silica nanospheres with OCP shell Firstly, 120 mg of the composite nanospheres were mixed with 30 mL silicon-saturated aqueous solution, forming a suspension of nanospheres, and the starting pH was adjusted to 6.5. It is assumed that use of silicon-saturation solutions would prevent dissolution and structural changes of the nanoparticles. Then the PAAS-assisted synthesis of OCP shell on the composite nanospheres (at a theoretical molar ratio of silica/OCP y:1, denoted as Silica@Znx@OCPy) was carried by dropwise addition of 12 mL of Ca(NO3)2 (20 mmol/L) and 9 mL of sodium phosphate solution containing NaH2PO4 (10 mmol/L) and Na2HPO4 (10 mmol/L) in the mixtures through a recently developed approach.39 While the solutions were added, a 20 lL of PAAS was also added to the mixture and the amount of PAAS used was equivalent to a theoretical monomeric unit concentration of 10 lmol/L. The resulting mixture was held at 378C with stirring for over 3 h (ageing time).

In vitro bioactive-ions release study The SGNs and Silica@Zn-x@OCPy of the powder samples (400 mg) were immersed in polyethylene bottles, respectively, with 200 mL of Tris buffer supplemented with 0.5 mmol/L KCl electrolyte (pH ¼ 7.25) under continuous vibrating (120 rpm) in a water bath at 378C. Part medium (5.0 mL) was removed with a syringe and centrifuged at the scheduled time within the initial 24 h. The powders were dispersed with same volume of fresh Tris buffer and added into the bottles. With a prolongation time of over 24 h, 10 mL of mediums were refreshed at an interval time of 12 h and the powders were also added into the bottles.

Surface and structure analysis Changes in zeta-potential of the nanoparticles before and after zinc adsorption were determined by using an

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Scheme 1. Schematic procedure used to generate core-shell-like Silica@Zn@OCP nanospheres assembled layer-by-layer. This simplified process, which is labeled as Silica@Zn@OCP, (i) starts from preprepared silica core and requires only one LbL assembly step, (ii) provides an zinc-ions-container material, (iii) avoids active zinc and silicon ions releasing quickly within a short stage, by capping porous OCP shell as a barrier tailoring by polyelectrolyte. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

electrophoresis apparatus (Zetasizer-3000HSA) in 0.5 mmol/L KCl background electrolyte solutions at a series given pH condition adjusted by dilute NH4OH and HCl. The zeta-potentials of bare SGNs were also determined in the ZnCl2 background electrolyte solution with a ZnCl2 concentration of 100, 1000, 2000, and 5000 lmol/L, respectively. Average of four distributions was obtained from measurements performed within 4 min after sample preparation and the average from them was reported as the final

result. The particle size and surface area analysis were performed by dynamic light scattering spectroscopy (DLS) (Marvin 2000) and N2 sorption analysis (BET method, Micromeritics Tristar 3000). Morphology and elemental compositions of the specimens before and after OCP coating were studied using transmission electron microscopy (TEM, JEM 2010) connected with energy-dispersive X-ray analysis (EDX). Phase compositions of the nanoparticles before and after immersion in Tris buffers were deter-

Figure 1. Zeta-potential of different particles as a function of pH in 0.5 mmol/L KCl solution (a) and zeta-potential of SGNs as a function of pH in the presence of zinc ions (b). Symbols in (a) represent the experimental data, and the lines represent correlation of the experimental data with the pH-dependent model. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] Journal of Biomedical Materials Research Part A

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TABLE I Zinc Concentration in the Remaining Solution and Adsorption Ratio with an Initial pH Condition of 8.4 for the Initial Two Cycles First Cycle

Second Cycle

System

Assembling (lmol/L)

Coating (lmol/L)

Adsorption Ratio (%)

Assembling (lmol/L)

Coating (lmol/L)

Adsorption Ratio (%)

Silica@Zn-80 Silica@Zn-400 Silica@Zn-2000

0.3 0.6 2.5

1.5 4.8 15.8

98.87 99.37 99.55

1.8 2.8 12.2

1.9 5.4 29.1

98.05 98.76 98.70

mined by FTIR spectroscopy (transmittance mode; Nicolet) and X-ray diffraction (XRD) (Rigaku D/max-rA).

Ion concentration analysis The free zinc concentrations in the assembling and coating residual supernatants were determined, respectively, with atomic adsorption spectrometer (HITACHI180-50), and the zinc content in the nanoparticles was calculated as the difference of concentrations in the initial and equilibrium solutions. The calcium, phosphorus, silicon, and zinc concentration in Tris buffers were measured by inductivelycoupled plasma atomic emission spectroscope (ICP, Vista AX), and pH values were measured by pH electrode (Leici).

RESULTS Effect of pH and concentration on the zinc ion assembly Zinc concentrations in remaining solution with an initial condition of pH 8.4 for the first two cycles are

shown in Table I. At this pH, the remaining zinc in assembling solution was significantly low (0.3–2.5 lmol/L), and it released partly into coating solution during silica coating (less than 1.0%). Simultaneously, increase of zinc concentrations and assembling/coating cycles implies a negligible change of the effective storage ratio, but the higher was the initial zinc concentration in assembling solution, the more zinc released into coating solution. By decreasing pH of assembling solution to the physiological condition (pH * 7.4), the residual zinc increased up to 5.7–186.3 lmol/L in assembling solution and more zinc ions returned the coating solution (data now shown). Secondly, it can be seen from Figure 1(a) that the adsorption of zinc ions changes the isoelectric point (IEP) of silica surface. In particular, the shift of IEP on 0.4 and 1 pH unit was observed for silica with adsorbed zinc ion in the initial zinc concentrations of 80 and 2000 lmol/L, suggesting that the significant modification of surface charge property resulted from electrostatic zinc adsorption. Furthermore, the zeta-potentials of SGNs were increased with increas-

Figure 2. XRD patterns (I) and FTIR spectra (II) of the Silica@Zn-2000@OCP40 nanocomposite system before (b) and after immersion in HCl-Tris buffers for 72 h (c) and 168 h (d), respectively. Data on bare SGNs (a) are shown as a reference. In (I), the OCP peaks are denoted by the asterisk (*), and HA peaks (d) by the cross symbol (#). In (II), the bands related to phosphate, particularly PO4, are denoted by asterisk symbol. Journal of Biomedical Materials Research Part A

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Figure 3. Transmission electron microscopy micrographs of the Silica@Zn-2000@OCP40 nanosystems (a, f, with inset EDX spectrum and particle size distribution) after ageing for 3 h (I) and 12 h (II) in comparison with the Silica@Zn-2000 nanospheres after the first adsorption/coating cycle (g, with inset face scanning EDX images and EDX spectrum), and face scanning EDX images obtained for silicon (b), zinc (c), calcium (d), and phosphorus (e) of the nanosystems. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4. Changes in zinc concentration of the composite systems in Tris buffers (a) and the schematic representation of the pH-initiating zinc ion release process (b); the error bars represent the standard deviation (n ¼ 3). Triangles: from Silica@Zn-2000@OCP35; inverted triangles: from Silica@Zn-2000@OCP40; solid circles: from Silica@Zn-80@OCP40. There are three categories of possible releasing behaviors for such core-shell architecture as the accumulating (Sch-2a), steady-state (Sch-2b), or declining (Sch-2c) concentration profile directly associated with the frequency of medium refreshing. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] Journal of Biomedical Materials Research Part A

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TABLE II Physicochemical Properties of the Silica@Zn-2000@OCP40 Particles Before and After Synthesis

Sample Silica Silica@Znb Silica@Zn@Silicac Silica@Zn@OCP OCP

Average Zeta-Potentiala (mV)

Average Size (nm)

BET Surface Area (m2/g)

52.1 45.1 51.8 24.2 10.6

78.9 80.1 88.5 98.4 136.5

94.3 92.9 78.9 148.1 –

a Average of three distributions obtained from measurements performed between 0 and 4 min after sample preparation in 0.5 mmol/L KCl background electrolyte solutions with a condition of pH 8.4. b For the silica nanospheres after adsorbing zinc ions for the first time. c For the composite nanospheres were LbL assembled alternately with the metal ions and silica for two cycles.

ing concentration of ZnCl2 background electrolytes at high pH in aqueous suspensions [Fig. 1(b)]. Positive zeta-potentials were determined under a pH condition between 6.6 and 8.4 in high zinc salt concentration of 5000 lmol/L. This result is possibly due to the positively charged silica surface anchored by a great amount of zinc ions at low pH (such as pH 6.6–7.2) and zinc hydroxide formation with increasing pH in aqueous suspensions (such as pH 7.8–8.4).

Silica@Zn-x@OCPy systems formation Figure 2 shows the powder XRD patterns and FTIR spectra of nanospheres before and after capping with OCP shells. Data on the Silica@Zn2000@OCP40 formulation are shown as a representative example. In contrast to low-intensity diffuse peak (*238/2h) of noncrystalline silica observed in bare SGNs [Fig. 2(Ia)], three additional peaks are detected in the composite nanospheres [Fig. 2(Ib)]. Specially, the main peak (4.828/2h) corresponded to the diffraction of (100) plane of OCP (d100 ¼ 18.68 ˚ ). The FTIR analysis also reveals that the spectra A exhibited a SiOSi stretch and SiOSi bend bands at *1030 and *470 cm1 for SGNs [Fig. 2(IIa)], and after capping with OCP shell, the bands at 1020–1090, *578 cm1 could be assigned to the phosphate group (PO4) for the Silica@Zn-2000@ OCP40 system [Fig. 2(IIb)]. Next, the morphology and microstructure of multilayer systems were established by TEM and EDX images in Figure 3. It can be seen that noticeably thin shells with nice contrasts (*5 nm) were obtained at an ageing time of 3 h [I, Fig. 3(f)]. FurJournal of Biomedical Materials Research Part A

ther, the shell appeared to grow with increasing ageing time [II, Fig. 3(f)] and its surface zeta-potentials (24.2 mV) was also much larger than that for SGNs (51.8 mV), but unequal to that of pure OCP (10.6 mV, Table II), implying an incompact coverage of nanospheres by OCP. The EDX images in Figure 3(b–e) clearly showed the distribution of silica core, zinc, and OCP shell in the nanospheres compared with those before OCP capping [Fig. 3(g), inset]. These desired outcomes reveal the anchor of zinc ions in silica core and coverage of silica core by OCP. Simultaneously, the EDX profiles [Fig. 3(a,g), inset] clearly demonstrated the difference in detectable compositional change of nanospheres. Zinc capacity was 0.70% in the Silica@Zn-2000 system in the first assembly cycle and subsequently of which increased up to 1.07% when the second assembly cycle and OCP capping were finished, confirming that increase of reproducible assembly cycles indeed increases the zinc storage capacity. A more striking feature is a significant shift on the surface area as well as particle size with surface modification (Table II). There appears to be significant increase on apparent specific surface area of nanospheres although the particles grew during capping OCP shell, suggesting that the porous OCP shells are successfully capped and tailored as porous structure. Additionally, the apparent ‘‘swelling’’ behavior of nanospheres before and after assembling/coating procedures was evident from the DLS analysis [Fig. 3(a), inset]. Particle sizes exhibit uniform size distribution and increase with each assembling/coating procedure, but no noticeable effect on broadening of size distribution.

In vitro controlled release behavior The changes in zinc concentration of Tris buffers are presented in Figure 4(a) during soaking Silica@Zn-x@OCPy systems (x ¼ 80 and 2000, x is the zinc ion concentration; y ¼ 35 and 40, y is the theoretical Silica-to-OCP molar ratio). Initially, there was a negligible amount of zinc ions leaching within the initial 2 h. Thereafter, low doses of zinc ions released from the Silica@Zn-x@OCPy systems and the increase in zinc concentration in response to Tris buffer mediums remained elevated at least 120 h. Notably, the zinc concentrations differed significantly among the systems with different zinc storage capacity and OCP shell thickness over an 8-h period. Relative to 0.12 ppm with the Silica@Zn-2000@OCP35 system after the initial 8 h of immersion, zinc concentration increased *0.32 ppm with Silica@Zn2000@OCP40, whereas that with Silica@Zn-80@OCP40 was only *0.05 ppm. Interestingly, for longer soak-

TAILORING MULTILAYER CORE-SHELL NANOSTRUCTURE

Figure 5. Changes in silicon concentration of three composite systems (a) in Tris buffers (average value in the three independent experiments, n ¼ 3); Data on bare SGNs (b) are shown as a reference. Triangles: Silica@ Zn-2000@OCP35; inverted triangles: Silica@Zn-2000@OCP40; solid circles: Silica@Zn-80@OCP40; open squares: bare SGNs.

ing times the zinc concentration reached a plateau and then maintained a zigzag-like sustained-release, although the mediums were partly refreshed periodically. These findings confirm a good storage and capping efficacy of silica/OCP multilayer on the encapsulated zinc ions.

Figure 6. Changes in pH (average value in three independent experiments, n ¼ 3) in Tris buffers during immersion of three composite systems, and data on bare SGNs are shown as a reference. Triangles: Silica@ Zn-2000@OCP35; inverted triangles: Silica@Zn-2000@OCP40; solid circles: Silica@Zn-80@OCP40; open squares: bare SGNs.

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Figure 5 shows the changes in silicon concentration with a modification of OCP shell. Compared with the Silica@Zn-2000@OCP35, the relative release rate (DSi concentration) of Silica@Zn-2000@OCP40 with thinner OCP shell increased by 20–35% over the initial 24 h (p < 0.05), but far slower than that with SGNs (130 ppm) within same time stage, suggesting that the thicker is the OCP shell on the silica-based nanospheres, slower the silicon release from the system. Figure 6 shows plots of pH value as a function of soaking time. A slow increase of pH occurred within the initial several hours. Subsequently, the pH generally increased corresponding to the elevated release rate of zinc ions via an ion-exchange mechanism with protons or background electrolytes. The pH in the solution soaking Silica@Zn-2000@OCP40 was remarkably higher compared with the solution soaking Silica@Zn-80@OCP40. This pH difference is also consistent with the zinc release patterns in the two systems [Fig. 4(a)]. Figure 7 indicates the release behavior of phosphorus and calcium in the systems. As can be seen, the OCP shell dissolved slowly in such phosphorus- and calcium-free buffer mediums and the changes in concentration of phosphorus and calcium followed a similar trend. With a prolongation of soaking time, the phosphorus and calcium concentration reach a plateau and then maintained an elevated- and decreased-release, respectively, when the mediums were partly refreshed. The XRD patterns and FTIR spectra for the immersed samples both gave specific peaks of new CaP, 32.188 and 25.768/2h [Fig. 2(Ic,d)],

Figure 7. Changes in calcium and phosphorus concentration of three composite systems in Tris buffers (average value in the three independent experiments, n ¼ 3). Triangles: Silica@Zn-2000@OCP35; inverted triangles: Silica@ Zn-2000@OCP40; solid circles: Silica@Zn-80@OCP40. Journal of Biomedical Materials Research Part A

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and two increased small bands at 566 and 603 cm1 [Fig. 2(IIc,d)] split from PO bending vibration in PO4 around *596 cm1 with a prolongation of soaking time, which is normally taken to indicate the presence of thermodynamically stable hydroxyapatite (HA) on the nanoparticles.

ble for pH-mediated zinc ions assembly-release processes:  SiOH$  SiO þ Hþ

ðIÞ

 SiOH þ Kþ $ SiOK þ Hþ

ðIIÞ

 SiO þ Zn2þ $  SiOZnþ ðassemblyÞ DISCUSSION The development of bioactive materials which deliver trace levels of biologically active matters capable of stimulating specific cell responses and promoting proliferation, differentiation, and gene expression has been a major goal of biomaterials design.40–42 To our knowledge, the optimum dose of bioavailable trace elements have dual advantages of a decrease in side reactions and a longer time window during which cells can accommodate extracellular microenvironment, allowing finer mediation of cell activity without compromising viability. Thus, the significance of controlling temporal gradients within short times and maintaining sustained-dosage for a prolonged time lies on the fact that this concept may overcome the negative effect by ‘‘burst’’ release of the multiple active ions from biomaterials in the exposed surroundings. This is also highly important to develop the new biomaterials for enhancing bone regeneration synergistically. Previously, zinc was administrated in the CaP bioceramics by controlling lattice substitution, whereas the capacity of which should be kept very low in case the contradictory results between bioactivity and solubility, due to the increased stability of CaP carrier by incorporation of zinc ions.23,24,30 In contrast, recent advance in lattice substitution of silicon in CaP bioceramics is opening up application possibility in which dissolution is enhanced at grain boundary, but the enhancement of dissolution is insufficient for activating gene expression and promoting bone regeneration.31,43–45 In the present study, zinc ions can be encapsulated into a multilayered silica system by using LbL assembly route and the zinc storage capacity can also be adjusted by the ion concentrations and reproducible cycles. The LbL assembly technique involving with silica-based materials, which is based on its reversible pH-response upon oppositely charged metal ions, is an effective approach to create well-defined internal microstructures and to develop novel trace-elements-delivery systems. As described earlier, silica gel is chosen as a reservoir because it does not only has silanol groups which localize zinc ions, but also possesses essential trace elemental silicon (as soluble silicate anion) for enhancement of bone regeneration. Following are the likely reaction steps which are possiJournal of Biomedical Materials Research Part A

ðIIIÞ

 SiOZnþ þ Hþ $ SiOH þ Zn2þ ðreleaseÞ ðIVÞ In this way, the zinc ions readily form electrostatic adsorption on SGNs. The increase in storage capacity with pH resulted from two possible mechanisms: (1) an increase in the number of sites available for zinc ion adsorption, because more of the adsorbed protons (Hþ) and Kþ ions on the silanol groups were dissociated, and (2) an increasingly more negatively charged and thus more electrostatically attractive kinetics to the metal ions from body solution.36,37 Indeed, the striking fact is that the silica surface affinity to zinc ions is reversible with respect to pH changes in the exposed surroundings. Consequently, the metastable zinc ions anchoring on silanol groups above physiological pH are able to release partially into a physiological pH of buffer by exchanging with protons and electrolytes from the medium [Fig. 4(b), Sch-1]. This in turns leads to a minor increase in pH at the solid/liquid interface areas, thus inhibiting zinc release rate through the enhancement of silica surface affinity (Fig. 6). On the other hand, selection of initial condition to achieve an optimum steady-state zinc concentration [Fig. 4(b), Sch-2b] needs to accurately monitor the concentration and frequency of medium refreshing. In the present study model, it is inevitable to a zinc accumulation (zigzag-like concentration) in a slow refreshing frequency [Fig. 4(b), Sch-2a], but this type of experimental model can minimize the factors except for the multilayered microstructure characteristics. In particular, the LbL microstructure and LbL distribution for zinc ion in the system limit ion diffusive rate. That is, the ordered silica/OCP multilayers have decreased permeability at the increased depth, and the zinc ions in the outer layer diffuse preferentially. Therefore, it is convenient to adjust LbL assembly cycles and a reproducible LbL assembly is favorable to increase storage capacity and zinc ion controlled-release. The second takes into consideration of the protective efficacy of OCP shell to silicon controlled-release. OCP is believed as a precursor of biological apatite in bones and can promote bone formation by releasing calcium and phosphate ions.46–48 It has been well documented that OCP morphogenesis can be regu-

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lated as porous microspheres by polyanionic biomolecules.39,49 According to our results, when Ca2þ- and PO43-contained salt titrants were added dropwise in the silica-containing suspension, a porous OCP shell are indeed coated on silica-based particles mediated by polypeptides, the nucleation mechanism of which may be similar to the metastable OCP formation on silica bioceramics in physiological environment.50 Apparently, the specific surface area of particles is reduced with particle growth if the particle surface is smooth or its porosity is less compared with the initial particle. In our study, the apparent specific surface areas of the nanospheres are decreased with the zinc assembling and silica coating, but appear actually to be a significant increase when coating OCP shells (Table II). This result suggests that, as expected, the OCP shell has higher porosity than the silica-based particles. Accordingly, it is favorable to combine advantages of tunable shell microstructures together with its thickness so that the desired temporal gradients within short times and maintaining sustained-dosage for a prolonged time can be achieved. Recently, with increasing knowledge of bone-cell response of bioactive glasses occurring in vivo, numerous in vitro studies have been devoted to the active ions release characteristics in buffer solutions. These biomaterials have been investigated that amounts of silicon dissolve at short times and the silica network degradation shows a significant dependence on chemical composition and textural properties and so on.25–29,51,52 These complexities have not only to be taken into account in short-term cellular events in vitro and in vivo, but also increase the difficulty in optimizing material bioactivity compared with the multilayered nanospheres. Moreover, the hydrolysis of OCP to HA has been suggested to take place in situ via phase transformation.47,48 Because the in situ transformation could produce phosphoric acid continuously from OCP, it may produce an acidic microenvironment, at least in the vicinity of the porous OCP shells, thereby modulating the milieu of the exposed silica core surface on which active ions exchange accelerate.

CONCLUSION We successfully explore a new method that allowed us to construct multilayer Silica@OCP nanosphere system that is potentially easier to control the biologically active ion storage capacity and release than the previous pathways. In this process, metal ions are LbL assembled and the rates of release are governed in response to stimulus-responsive consequence, and porous OCP shell formation rate is controlled by degree of solution supersaturation with

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respect to OCP nucleation and ageing time. An important aspect of this study is that it presents a new example of the potential biomedical application of biologically active trace elements encapsulation. Considering that controlled-release of the system can be easily attained with appropriate assembly approaches, the extension of the presented technique to other multiple trace elements encapsulation will enable the preparation of a variety of biomaterials with different functionalities for fundamental study and utilization in bone regeneration and repair. The authors appreciate the kind help of Dr. J. Liang, Mr. C. Wang, and Mr. J. Zhao for experimental assistance in BET, ICP, and DLS measurements of the materials and helpful discussions.

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