A molecular investigation of the initial nucleation of calcium phosphate

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A molecular investigation of the initial nucleation of calcium phosphate on TiO2 substrate: the effects of surface nano-topographies Ting Zheng, Chunya Wu, Yu Zhang, Mingjun Chen, and Peter T. Cummings Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b01546 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 7, 2018

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Crystal Growth & Design

A molecular investigation of the initial nucleation of calcium phosphate on TiO2 substrate: the effects of surface nano-topographies Ting Zheng*†‡, Chunya Wu*‡, Yu Zhang§, Mingjun Chen‡, Peter T. Cummings§

†

Department of Mechanical Engineering, Dalian Maritime University, Dalian 116026, P. R. China

‡

State Key Laboratory of Robotics and System, and School of Mechatronics Engineering, Harbin Institute of Technology, Harbin 150001, P. R. China

§

Department of Chemical and Biomolecular Engineering, and Multiscale Modeling and Simulation Center, Vanderbilt University, Nashville, Tennessee 37235-1604, United States

* Corresponding author: Ting Zheng Chunya Wu

Email address: [email protected] Email address: [email protected]

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Abstract Nucleation and biomineralization of apatite on titanium-based material surfaces is crucial to improve surface biocompatibility, osseointegration and rapid bone ingrowth in biomedical applications such as joint replacements. This work is designed to provide new insights on the molecular processes in the initial nucleation of calcium phosphate on TiO2 surface by means of classical molecular dynamics (MD) simulations. Aggregation of calcium and phosphate ions in pure aqueous solution was studied and the free energies during ion adsorption was investigated by the calculation of PMFs. The MD results suggest surface hydroxylation rate and nano-topographies of TiO2 substrate contribute significantly to the initial nucleation of calcium phosphate. Our simulations suggest that surface hydroxyls on TiO2 provide active sites for the aggregation of calcium phosphate. Both calcium ions and phosphate ions could bind to the hydroxylated TiO2 surface directly or indirectly via the 1st water layer. Surface nano-topographies (e.g., grooves or ridges) seem to be able to restrict the diffusion of calcium ions and phosphate ions, hence offering more opportunities for adsorption by trapping ions inside channels. Therefore, it can be inferred that apatite may be formed more favorably on porous or concave surfaces.


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Introduction Titanium-based materials are commonly employed in biomedical devices and components, especially as hard tissue replacements 1 . Exceptional osseointegration, interpreted as direct access of bone tissues onto Ti-based biomaterials, is quite essential for the long-term success of therapy 2 . Therefore, various surface modification techniques have been conceived and tested to achieve excellent bone-biomaterials interactions3. Calcium and phosphate ions are among the main components presented in body fluid, and also comprise the main inorganic phase of human bones4.Formation of bonelike apatite on implant surface has been proven to be extraordinary to improve bone bonding5. Ti-based biomaterials coated with bonelike apatite can be obtained by various methods, such as sol-gel deposition, chemical vapor deposition (CVD), thermal spray or plasma spray6. These surface preparations assist osteoblast cells to bind to the surface apatite layer, differentiating actively to form bone-like tissue7,8. Kokubo9 proposed that the ability of apatite formation on the biomaterial surface in simulated body fluid (SBF) was very promising for predicting the in vivo bone bioactivity. Therefore, the ability and the underlying mechanism of the formation of apatite on the biomaterial surface in solution have been raised great attention. The hydrated titania surface, vulnerable to the attack of hydroxyls in solution, is predisposed to produce the negatively charged hydrated species (HTiO3−) and attract Ca2+ ions in aqueous solution, which will eventually leads to the supersaturation of calcium and phosphate ions near the surface10. Most obviously, the micro- and nano-topographies on the surface, generated by



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chemical, physical or mechanical approaches, have been demonstrated as significant factors affecting the biological behaviors of cells (e.g., differentiation, proliferation and orientation)11. Meanwhile, the presence of surface nano-topographical features also helps to stimulate the initial formation of apatite12,13,14. Aparicio15 revealed that apatite nucleation preferred to occur at the concave part of micro-roughened titanium plates, where the negative surface charges were presented at high density. Porous titanium or nano-structured surfaces also showed promising advantages for the bone-bonding performance16, and the porous structures may lead to an increase in surface area and surface energy, allowing higher levels of bone ingrowth 17 . As demonstrated by Matthieu18, the spatial gap size of the porous titanium scaffolds was an important topographical factor influencing the deposition of apatite, which would be induced more easily by the scaffolds with spatial gaps ranging from 200 to 700 µm. Wu19 prepared a nanostructured titanate layer on a Ti substrate and found that the layer favored the rapid deposition of hydroxyapatite by displaying super-hydrophilicity. Bsatet12 found that nano-topographical features within the size range of 200–300 nm on the surface of AlAcH-treated Ti6Al4V alloys were ideal for apatite formation. However, to the best of our knowledge, the underlying molecular mechanism of apatite

nucleation

on

the

Ti-based

substrate

in

the

presence

of

surface

nano-topographies has not been fully investigated. In this work, the interfacial interaction between calcium phosphate ions and TiO2 surface was investigated by MD simulations. The binding affinities of calcium ions and phosphate ions (abbreviated as Ca-P ions) on TiO2 substrate surface were evaluated by the calculation of potentials of


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mean force (PMF), which can yield a quantitative picture of the adsorption dynamics of ions. Special attention was paid to the aggregation of Ca-P ions on TiO2 surfaces characterized by nano-scale topographies.

Simulation Method The excellent surface properties (e.g., good corrosion resistance and biocompatibility) of Ti-based materials largely depend on a thin but strongly adherent layer of titanium dioxide, which is formed naturally in aerobic environment20. Since rutile TiO2 is the most stable crystal structure of titanium dioxide, the rutile TiO2 (110) surface is selected in this work to evaluate the effects of surface nano-structures on the initial nucleation of calcium phosphate. Atomic steps and grooves were artificially created by removing the —

TiO2 units along the crystal direction as described in our previous work21. The point of zero charge (pzc) of TiO2 is about 5.30 at 35 °C, based on which a fully hydroxylated neutral rutile TiO2 (110) surface was set up22. The forcefield parameters for the surface hydroxyls were obtained from Předota’s work23 or derived from the ab initio calculations24. In total, 5 kinds of TiO2 substrates, e.g., non-/fully-hydroxylated perfect TiO2 surface and the step-/groove-/ridge-structured TiO2 surface with full hydroxylation, denoted as NP, HP, S, G and R respectively, were created and the surface characteristics are listed in Table 1. Both sides of the TiO2 slabs were designed with identical surface properties, including hydroxylation states and nano-structures, in order to average the interaction results from two separate interfaces. The forms of aqueous phosphate vary widely according to the pH value of solution, such as phosphate ions (PO43-, pKa= 12.32), hydrogen-phosphate ions (HPO42-, pKa=



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7.21) and dehydrogen-phosphate ions (H2PO4-, pKa= 2.16). Therefore, at pH= 5.30, when the TiO2 surface is electrically neutral, the main forms of phosphate ions are HPO42- and H2PO4-. The partial charges and parameters for Van der Waals interactions of HPO42-, H2PO4- and Ca2+ ions were taken from Yang’s theoretical study25, which are also listed in Table 2. The intramolecular structures of phosphate ions were described by the Amber force field26 and the SPC/E model27 was chosen for water molecules.

Table 1. Surface characteristics of different TiO2 substrate Items NP

HP

Substrate names

Conformations

Non-hydroxylated

Box Size (a×b×c) 4.28×5.38×6.55 nm3

perfect TiO2 surface Fully-hydroxylated

4.28×5.38×6.70 nm3

perfect TiO2 surface Step-structured TiO2

S

4.28×5.38×6.65 nm3

surface with full hydroxylation Groove-structured TiO2

G

4.28×5.38×8.16 nm3

surface with full hydroxylation Ridge-structured TiO2

R

4.28×5.38×8.06 nm3

surface with full hydroxylation

All simulations were performed with the Gromacs 4.5.5 code28. Aggregation of HPO42-, H2PO4- and Ca2+ in aqueous solution was conducted in NPT ensembles at P= 1 bar and T= 310.15 K, controlled by the Parrinello-Rahman approach 29 and the Nosé-Hoover thermostat30,31, respectively. Nucleation of Ca-P ions on TiO2 surface was


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conducted in NVT ensembles for 15 ns, during which similar strategies of determining the box size in z direction were chosen as described in our previous work32. The short-range electrostatic interactions were truncated at 1.2 nm, whereas the long-range interactions were calculated by the Particle-Mesh Eward (PME) method33,34.

Table 2. Atomic parameters for HPO42- and H2PO4- ions Atomic charge (e) Nomenclature of atoms HPO42(pKa= 7.21) H2PO4(pKa= 2.16)

VdW parameters

HPO42-

H2PO4-

σ (nm)

ε (kcal/mol)

OPH

-0.73

-0.63

0.354

0.1521

OP

-0.90

-0.72

0.34

0.12

P

1.10

1.04

0.43

0.585

H

0.33

0.33

0.0449

0.046

The adsorption strength and free energies of Ca-P ions on TiO2 surface were estimated by the potentials of mean force (PMF) calculations. The position-dependent PMF could be extracted using umbrella sampling through a series of MD simulations, where the z-distance from the center of mass (COM) of ions (Ca2+ or HPO42-) to the first Ti layer was considered as the reaction coordinate. A total of 27 configurations was generated along the reaction coordinate for each case and the space between two adjacent windows ranged from 0.02 nm to 0.1 nm. A harmonic potential with a spring constant of k= 1000 kJ·mol-1 was exerted on the COM of ions to conduct the umbrella sampling. Each configuration was sampled for 9.5 ns after a short equilibration process of 0.5 ns. The PMF profiles were extracted by the weighted histogram analysis method (WHAM)35.



Results and Discussion 3.1 Aggregation of calcium phosphate ions in aqueous solution The free aggregation of Ca-P ions in aqueous solution was studied first. The MD simulation, containing 25 HPO42-, 25 H2PO4-, 75 Ca2+ and 75 Cl- ions in a box with a dimension of 5×5×5 nm3, was conducted under the NPT ensembles for 6 ns. Hydrated water shells are formed around the Ca2+, HPO42- and H2PO4- ions during the simulation. For the Ca2+–H2O interaction, the first peak in the radial distribution functions (RDFs) of Ca2+–OW centers at 0.24 nm (Figure 1a), corresponding to the location of water oxygen (OW) atoms in the first hydration shell around Ca2+ ions. The first hydration shell with respect to Ca2+ ions contains almost 8 water molecules, consistent with previously reported results36. The RDFs of phosphorus atoms (P) in HPO42- and H2PO4- ions with respect to OW atoms are shown in Figure 1b and Figure 1c, respectively. The RDFs of P–OW exhibits the first peak at 0.38 nm and 0.382 nm, respectively, which are comparable to the data reported in de Leeuw’s work37 (0.373 nm and 0.376 nm for HPO42- and H2PO4- ions). The calculated coordination numbers (CN) of OW atoms around HPO42- and H2PO4- ions are 13.8 and 11.4, respectively. Combined with the analysis of the conformations as also shown in Figure 1, we deduce that the numbers of water molecules in the hydration shell around the HPO42- and H2PO4- ions are 12~14 and 11~13, respectively. The difference in hydrated shells around HPO42- and H2PO4- ions may result from the protonation of H2PO4- ions, which repels water molecules more strongly than HPO42- ions. De Leeuw37 also reported that the strength of the H-bond between OP atoms and the surrounding water hydrogens (HW)


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decreased as the oxygen site became protonated.

Figure 1. RDFs between ions (based on Ca or P atoms) and OW atoms, as well as the corresponding coordination number. a) Ca2+-OW, b) HPO42--OW and c) H2PO4--OW

Calcium ions coordinate monodentately or bidentately to the oxygen atoms (OP or OPH) in HPO42- or H2PO4- ions via the electrostatic attraction, which accords well with the structural conformations of Ca2+–PO43- clusters described by de Leeuw38. The first peak in the RDFs of Ca2+–OP centers at 0.246 nm (shown in Figure S1 in the Supporting Information) as a result of the strong electrostatic attraction. The amplitudes of the first peaks in RDF curves of Ca2+–OP1 and Ca2+–OP2 (OP atoms in HPO42- and H2PO4- ions respectively), denoted by gmax, are compared with each other to distinguish 2+

2+

Ca -OP1 Ca -OP2 their binding affinities. The value of g max is 17.9, indicating that / g max

calcium ions are much more favored at the HPO42- sites. 3.2 Adsorption of ions on rutile TiO2 (110) surface Deposition of Ca-P ions on the surface of Ti-based biomaterials appears to be able to endow the surface with favorable bioactivity5,9. However, the dynamic process of the bio-mineralization on Ti-based biomaterials is still unclear. In this section, adsorption of Ca-P ions on nanostructured rutile TiO2 (110) surfaces, is studied to investigate the



initial nucleation of apatite. Adsorption systems with 30 HPO42-, 30 H2PO4-, 60 Ca2+ ions and 30 Cl- ions on TiO2 substrate were set up and performed to run under NVT ensembles for 15 ns. The number densities of ions as a function of the distance from the surface Ti atom layer of NP- and HP-TiO2 substrates along the z direction are shown in Figure 2a and 2b. RDFs between HP-TiO2 surface hydroxyls (based on oxygen atoms of the surface terminal hydroxyl groups, OTH) and ions (based on Ca and P atoms, which are represented by P1H and P2H for HPO42- or H2PO4- ions respectively) are shown in Figure 2c. On the NP TiO2 surface, both calcium ions and phosphate ions distributed mainly beyond the 2nd water layer (as shown in Figure 2a), suggesting an unfavorable nucleation of Ca-P ions on the NP-TiO2 surface. The surface hydroxyls on the HP-TiO2 (110) surface, however, seem attractive to Ca-P ions. Compared with 1st and 2nd dense water layers on the NP-TiO2 surface, water molecules are less structured with a peak value at z=0.375 nm from the surface Ti atom layer as a result of surface hydroxyls(Figure 2b). The number density of Ca2+ ions exhibits its 1st peak at 0.375 nm, indicating a direct interaction between Ca2+ and surface hydroxyls. Therefore, the loosely patterned water molecules on HP-TiO2 surface may be inferred as much smaller barriers for ions to break through. The distribution of Ca2+ shows a second peak at z=0.509 nm following (after the peak of phosphate ion); this second peak has a larger amplitude than the first peak as a result of the structured water layer, which will be discussed in detail in the following section. As shown in Figure 2b, the distribution of HPO42- ions along the z direction shows


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the 1st maximum at 0.476 nm, interacting with surface hydroxyls via hydrogen bonds as illustrated by the local conformations in Figure 2c. Adsorption of HPO42- on TiO2 surface is much stronger than H2PO4-, as inferred from the larger values in the 1st RDF peak. Thus, the increased protonation of phosphate ions may result in the decrease of binding affinity of ions on the HP-TiO2 surface.

Figure 2. Number densities of Ca-P ions on the NP- and HP-TiO2 surface (a and b respectively), as well as the RDFs between Ca-P ions and the surface hydroxyls on the HP-TiO2 surface c).

In order to quantitatively evaluate the binding affinities of different ions (Ca2+ and HPO42-) on the HP-TiO2 surface, the free energy during ion adsorption was studied by the calculation of PMFs. As shown in Figure 3a, the distribution of PMF curves during the adsorption of Ca2+ ions exhibits four minima (indicated as 1~4). The 1st minimum locates almost at the same position of 1st water layer (marked as position A in Figure 3 at r=0.375 nm), with a binding energy of 14.0 kJ/mol. Figure 3b displays the adsorption conformation of Ca2+ ions and it can be clearly seen that the Ca2+ ions bind stably with the adjacent two terminal hydroxyls (TOH). The 3rd minimum locates between the



centers of the 1st (position A) and the 2nd water layer (marked as position B) with a binding energy of 7.5 kJ/mol. The corresponding conformation of Ca2+ ion via the mediation of hydrated water shells, interpreted as hydrated adsorption sites, is shown in Figure 3c. As indicated in the PMF curves, a large energy barrier of 13.8 kJ/mol needs to be overcome for Ca2+ ions transferring from this hydrated adsorption site (minimum 3) to the direct adsorption site (minimum 1). Therefore, Ca2+ ions may remain relatively stable at the hydrated adsorption sites, which may explain why Ca2+ ions show a much higher number density value at the 2nd peak than the 1st peak in Figure 2b. The 4th minimum in the PMF curve appears after the 2nd water layer (position B) on the HP-TiO2 surface. Consequently, Ca2+ ions at this position move more freely since the water is not strongly structured in this distance. We also note the 2nd minimum in the PMF curves; however, no stable conformation corresponding to this minimum could be extracted from the trajectory. Based on the analysis of the dynamic adsorption conformations, Ca2+ ions at the hydrated sites may reach minimum 2 and stay for a very short time after failing to break through the layered water barriers to reach the direct binding sites.

Figure 3. PMF profile during the adsorption of Ca2+ on the HP-TiO2 surface a), as well as the direct adsorption conformations b) and the hydrated adsorption conformations c).


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The free energy during the adsorption of HPO42- ions was also studied by the calculation of PMFs, as shown in Figure 4. The z-distance between the center of mass (COM) of the HPO42- ions and the surface Ti atom layer is chosen as the reaction coordinate (r). The HPO42- ions located at the 1st minimum can interact with the surface hydroxyls directly with a binding energy of 15.7 kJ/mol (the corresponding adsorption conformation is shown in Figure 2c), whereas the minimum 2 in the PMF curve could be inferred as an indirect adsorption mode of HPO42- ions via the 1st layer water molecules (conformations not shown here). Nevertheless, the energy barrier for HPO42- ions at the minimum 2 to move either towards the direct binding sites at minimum 1 or back to bulk water is only ~5 kJ/mol. Compared with the adsorption of Ca2+ ions, it is easier for HPO42- ions to reach the surface hydroxyls on the HP-TiO2 substrate. As inferred from the small peak value in the RDFs of H2PO4- ions in Figure 2b, adsorption of H2PO4- ions is less stable than HPO42- ions. Therefore, in this case, the free energy during the adsorption of H2PO4- on the HP-TiO2 surface was not investigated.

5

3.5 3

A

2.5

-5

2

-10

Water Density

B

-15

2

PMF

1.5 1

Density (g/cm3)

0 PMF (kJ/mol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.5

1

-20

0 0

0.5

r (nm)

1

1.5

Figure 4. PMF profile during the adsorption of HPO42- ions on the HP-TiO2 surface.



In order to investigate the effects of surface nanostructures on the aggregation of Ca-P ions, MD simulations of the adsorption of Ca-P ions on perfect TiO2 surface (NP/HP), as well as on the fully hydroxylated surfaces with nano-topographies such as steps (S), grooves (G) and ridges (R), were conducted in NVT ensembles for 50 ns. The number densities of Ca-P ions (based on Ca2+ and P atoms) on TiO2 surfaces within the first and final 5 ns (0-5 ns and 45-50 ns, respectively) were analyzed and compared (Figure 5).

Figure 5. Number densities of Ca-P ions in the first and final 5 ns on a) non-hydroxylated perfect (NP) TiO2 surface, b) fully-hydroxylated perfect (HP) TiO2 surface, c) step-structured (S) TiO2 surfaces, d) groove-structured (G) TiO2 surfaces and e) ridge-structured (R) TiO2 surfaces.

Results for the aggregation of Ca-P ions on the NP- and HP-TiO2 surface are shown in Figure 5a and 5b. Distribution of Ca-P ions on the NP-TiO2 surface mainly concentrated beyond the 2nd water layer, ranged from 0.45 nm to 1.0 nm. However,


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favorable accumulation of Ca-P ions was observed on the HP TiO2 surface (Figure 5b) with the concentrated distribution ranging from 0.2 nm to 0.4 nm from surface Ti atom layer. The similar phenomenon was observed by Ohtsuki39 that the adsorption of Ca-P ions from the SBF was promoted on TiO2 surface prepared by the sol-gel method, where substantial hydroxylation is exhibited. The outlines of the distributions of number densities within the first and final 5 ns almost remain the same under these two conditions, which can be inferred as limited accumulation of Ca-P ions on perfect TiO2 surface, especially on the NP-TiO2 surface. The accumulation of Ca-P ions on the step-structured TiO2 surface (S) is enhanced as indicated by the increased value of the first peak in the final 5 ns period in Figure 5c. The step edges on TiO2 surface could provide more adsorption sites for Ca-P ions by exposing more hydroxyls (conformations not shown here). Moreover, compared to the results of the first 5ns, the outline of number densities in the final 5 ns changes slightly after the 1st peak, which may be attributed to the limited dimensions of the step structures. After extending the surface step-structures to grooves and ridges on TiO2 substrate (G and R), the adsorption behaviors of Ca-P ions at the presented two time periods seems rather different. The distributions of number densities of Ca-P ions on grooved-structured and ridged-structured TiO2 surface both exhibit two peaks at r=0.22 nm and r=0.5~0.62 nm as shown in Figure 5d and 5e. The corresponding adsorption conformations of Ca-P ions on the ridge-structured TiO2 surface at t= 50 ns are shown in Figure 6a. Ca-P ions interacting with the surface hydroxyls directly are highlighted



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by the dashed green rectangle. However, Ca-P ions in the second neighborhood of the substrate surface (0.45 nm

A molecular investigation of the initial nucleation of calcium phosphate

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