Sensing Ability of Orthopedic Implant Sensors

Of course, the next question is how well do these MWCNTs sense new bone growth? As a starting point, experiments were conducted with the Fe(CN)4-/3-redox system. The Fe(CN)4-/3- redox system with a heterogeneous one electron transfer (n = 1) is one of the most extensively studied redox couple in electrochemistry (Tamir et al. 2007). It has been performed on cyclic voltammetry experiments (the Fe2+/3+ redox couple) by placing MWCNT-Ti in an electrolyte solution of 10 mM K3Fe(CN)6 and 1 M KNO3. In potassium ferricyanide (K3Fe(CN)6), the reduction process is Fe3+ (Fe(CN)3- + e" ^ Fe(CN)4-) followed by the oxidation of Fe2+ (Fe(CN)4- ^ Fe(CN)3- + e-) under a sweeping voltage. In this study, the iron (II/III) redox couple did not exhibit any observable peaks for bare Ti or anodized Ti electrodes, as shown in Fig. 7a, b. This implied that the electrochemical reaction was slow on both of these electrodes. However, highly directed electron transfer at the MWCNT-Ti sensor electrode was observed as redox peaks, shown in Fig. 7c. At a scan rate of 100 mV/s in Fig. 7c, a well-defined redox peak appears with anodic

b cd

QjJ 2

QjJ 2

Anodized Ti

MWCNTs grown on Anodized Ti

Carbon Nanopaper

Fig. 4 Osteoblasts long-term functions: (a) Alkaline phosphatase activity; values are mean ± S.E.M; n = 3; *p < 0.05 compared to Ti; **p < 0.1 compared to anodized Ti; and #p < 0.1 compared to carbon nanopaper and (b) calcium deposition; values are mean ± S.E.M; n = 3; *p < 0.1 compared to Ti; **p < 0.1 compared to anodized Ti; and #p < 0.05 compared to carbon nanopaper

Anodized Ti

MWCNTs grown on Anodized Ti

Carbon Nanopaper

Fig. 4 Osteoblasts long-term functions: (a) Alkaline phosphatase activity; values are mean ± S.E.M; n = 3; *p < 0.05 compared to Ti; **p < 0.1 compared to anodized Ti; and #p < 0.1 compared to carbon nanopaper and (b) calcium deposition; values are mean ± S.E.M; n = 3; *p < 0.1 compared to Ti; **p < 0.1 compared to anodized Ti; and #p < 0.05 compared to carbon nanopaper b

(E ) and cathodic (E ) potentials at 175 and 345 mV, respectively. Moreover, as shown in Fig. 7e and 9c, the relationship was linear between the anodic and cathodic peak currents versus the square root of the scan rate, while the ratio of I /I was about 1, corresponding to the Randles-Sevcik equation (1). Because the root scan rate had this linear relation with the peak currents, the mass transport in this process must be by diffusion. Zhang et al. found that the heterogeneous chargetransfer rate constant (k) of the Fe(CN)4- /3- complex with H2O as a solvent was 0.05 cm/s (Zhang et al. 1991). Since the k value was in the range of 10-4 to 10-1 cm/s and DEp > 59/n mV (in this case n = 1 and DEp ~ 170 mV), this process was quasireversible.

To analyze the electrochemical behavior at the surface of the MWCNT-Ti sensor electrodes, researchers have used the Randles-Sevcik equation for quasireversible processes, equation (1). Hence, the peak current (I) is given by:

where n is the number of electrons participating in the redox process, na is the number of electrons participating in the charge-transfer step, A is the area of the working electrode (cm2), D is the diffusion coefficient of the molecules in the electrolyte solution (cm2/s), C is the concentration of the probe molecule in the bulk solution (molar), and Y is the scan rate of the sweep potential (V/s). When the Fe(CN)4-/3-redox system exhibits heterogeneous one-electron transfer (n = na = 1) and the concentration C is equal to 10 mM, the diffusion coefficient D is equal to 6.7 ± 0.02 x 10-6 cm2/s (Hrapovic et al. 2004; Hrapovic and Luong 2003). Hence, the quasireversible redox of iron (II/III) is truly enhanced by the novel MWCNT sensors.

To estimate how such sensors would do for bone growth, osteoblasts were cultured for 21 days and energy dispersive spectroscopy (EDS) was performed to verify the presence of the various minerals in newly formed bone. Figure 5 shows the results of one such study doing this in which the peaks of many inorganic substances, consisting of magnesium (Mg), phosphorus (P), sulfur (S), potassium (K), and calcium (Ca), were detected. The Ca/P weight ratio of minerals deposited by osteoblasts in that study on Ti (1.34) was less than that on a MWCNT-Ti sensor (1.52). However, the Ca/P ratio of hydroxyapatite (HA), the main calcium-phosphate crystallite in bone, is typically about 1.67 (Calafiori et al. 2007; Wang et al. 2003). This study demonstrated that the minerals deposited by osteoblasts on MWCNT-Ti were more similar to natural bone than the minerals deposited on Ti. X-ray diffraction (XRD) analysis also showed that more hydroxyapatite was deposited on MWCNT-Ti sensors than on both conventional and anodized Ti after 21 days of culture, as shown in Fig. 6. In addition, the amount of calcium deposited by osteoblasts as determined by a calcium quantification assay kit was 1.481 mg/ cm2 for 7 days, 1.597 mg/cm2 for 14 days, and 2.483 mg/cm2 for 21 days on conventional Ti, as shown in Fig. 10b. These results imply a greater deposition of calcium by osteoblasts on MWCNT-Ti sensors than currently implanted Ti.

Bone resorption and remodeling involve the secretion of hydrochloric acid (HCl) by osteoclasts (Zaidi et al. 2004). Osteoclasts dissolve bone mineral by isolating a region of the matrix and then secreting HCl and proteinases at the bone surface, resulting in the bone acting as a reservoir of Ca2 +, PO4- , and OH- minerals (Blair et al. 2002). This can be the reason that HCl is so prevalent in laboratories to dissolve calcium minerals deposited by osteoblasts on scaffolds in vitro (as the supernatant to further use in a calcium deposition assays). Thus, after dissolving calcium mineral with 0.6 N HCl, it is likely that Ca2+, PO;j- , and OH- are contained in the solution of the osteoblast extracellular components.

The formation of bone matrix minerals first depends on achieving a critical concentration of calcium and phosphorus. Then phospholipids, anionic proteins, as

Elements

Weight%

Atomic%

C

13.16 ± 5.73

21.65

O

45.83 ± 3.09

56.62

Mg

0.20 ± 0.07

0.16

P

13.29 ± 0.91

8.48

S

1.27 ± 0.14

0.78

K

0.29 ± 0.09

0.15

Ca

17.86 ± 1.21

8.81

Ti

8.11 ± 0.58

3.35

Co

0.00 ± 0.00

Co Mg ca

Ii

Elements

Weight%

Atomic%

C

64.22 ± 0.70

74.79

O

23.78 ± 0.57

20.79

Mg

0.12 ± 0.3

0.07

P

3.70 ± 0.09

1.67

S

0.06 ± 0.03

0.03

K

0.14 ± 0.02

0.05

Ca

5.62 ± 0.12

1.96

Ti

1.54 ± 0.08

0.45

Co

0.81 ± 0.06

0.19

Energy (keV)

Fig. 5 EDS analysis of osteoblasts cultured for 21 days on (a) Ti and (b) MWCNT-Ti. SEM micrograph of inset (b) shows the analyzed area. For the MWCNT-Ti, more Ca and P deposited by osteoblasts were observed. Tables in (a) and (b) show the composition of the mineral deposits after osteoblasts were cultured for 21 days. The Ca/P weight ratio on bare Ti was 1.32 and on MWCNT-Ti was 1.52

a b well as calcium and phosphorus aggregate in nucleation pores that are in the 35 nm "hole-zone" between collagen molecules (Bronner and Farach-Carson 2003). The addition of calcium, phosphate, and hydroxyl ions contributes to the growth of crystalline hydroxyapatite. However, the crystals are not pure since they also contain carbonate, sodium, potassium, citrate, and traces of other elements, such as strontium and lead. The imperfection of hydroxyapatite contributes to its minerals' solubility, which plays a role in the ability of osteoclasts to resorb the mineral phases. Although osteoblasts synthesize type I collagen, which is the predominant organic components of bone, type III/V/VI collagen also exist in bone. Moreover, noncollagenous proteins in bone (such as growth factors, osteocalcin, osteopontin, and osteonectin) and proteins in serum and other tissues (such as fibronectin, vitronectin, and laminin) are absorbed into the mineral component during bone growth. These components are directly involved in the genesis and maintenance of bone. Thus, not only minerals, but also other noncollagenous proteins, are broken

Fig. 6 XRD analysis of hydroxyapatite-like (HA; Ca5(PO4)3OH) deposited minerals after osteoblasts were cultured for 21 days on (a) Ti and (b) MWCNT-Ti. The micrographs show that the peak pattern of HA more closely matches that of the mineral deposited by osteoblasts when cultured on MWCNT-Ti than Ti

2-Theta-Scale

Fig. 6 XRD analysis of hydroxyapatite-like (HA; Ca5(PO4)3OH) deposited minerals after osteoblasts were cultured for 21 days on (a) Ti and (b) MWCNT-Ti. The micrographs show that the peak pattern of HA more closely matches that of the mineral deposited by osteoblasts when cultured on MWCNT-Ti than Ti down after dissolution with HCl. As such, nonspecific proteins may exist in the supernatant of bone formation when cyclic voltammetry is performed on MWCNT-Ti sensors and may contribute to the observed redox process shown in this chapter. On an electrode surface, the native conformation of a protein may be retained

(reversible or diffusion-controlled) or distorted (irreversible or adsorption-controlled), depending on the extent of the interactions (such as the electrostatic or covalent bonds) between them.

The type of electrolyte is important to the redox reaction because the gained capacitance and scan rate window are dependent on it (Hong et al. 2002). Studies to date have not degassed with nitrogen or argon when performing cyclic voltam-metry with MWCNT sensors, thus, the redox reactions in CV are related to the H2O decomposition, which includes H+ reduction and OH- oxidation. The pH of the electrolyte also affects the H2O decomposition. In the case of an acidic 1 N HCl solution (without other electroactive molecules), the H+ ions induced the formation of H2 gas at -0.2 V as a result of the increased reduction current (Hong et al. 2002). In the alkaline 1 M KOH solution, OH- ions resulted in the formation of O2 gas at 0.7 V by the oxidation process. This fact should be noted because in vivo, these decompositions may occur at the interface of the Ti implant due to pH changes and the presence of H2O and O2 around the implant.

Other studies have shown promising results when using CNT-modified electrodes in biosensing. Since MWCNTs have good electrochemical characteristics as electron mediators and adsorption matrices (Sotiropoulou et al. 2003), they may further enhance applications in biosensor systems. For example, Harrison et al. suggested that CNTs offer a promising method to enhance detection sensitivity because they have high signal-to-noise ratios (Harrison and Atala 2007). The structure-dependent metallic character of CNTs should allow them to promote electron-transfer reactions for redox processes, which can provide the foundation for unique biochemical sensing systems at low overpotentials (Roy et al. 2006). The electrolyte-electrode interface barriers are reduced by CNTs because they facilitate double-layer-effects (Fang et al. 2006). Typically, when the supporting electrolyte is at least a 100-fold greater than the active electrolyte (Christian 1980), the charge in the electrolyte solution causes the Debye layer to be more compact. Therefore, this compact layer can rapidly exchange electrons between electroactive proteins and the surface of an MWCNT-Ti electrode. This is likely the reason the sharpened cathodic and anodic peaks in CV have been observed, as shown in Figs. 7c, 8c, 9b.

Although the area of the working electrode is constant in the presence of MWCNTs, the overall surface area and the electroactivity of the surface increases. The MWCNTs act as a nanobarrier between the titanium oxide layer, which was its growth template, and the electrolyte solution. The surface atoms or molecules of MWCNTs play an important role in determining its bulk properties due to their nanosize effects (Cao 2004). A large surface, corresponding to the greater electrocata-lytic activity, confers the catalytic role on the MWCNTs in the chemical reaction. Well-defined and persistent redox peaks are shown in Fig. 7c, confirming the increases in electron transfers and higher electrochemical activities at the MWCNT-Ti surface. Cyclic voltammetry was also performed with a glassy carbon electrode (GCE; Bioanalytical) and a platinum electrode (PTE; Bioanalytical), and it also showed DEp > 59/n mV (data not shown). Importantly, for the same scan rate, the CV of GCE and PTE showed redox reactions with similar but more widely separated anodic and cathodic peaks, confirming the performance of the MWCNT-Ti electrode.

Potential (mV) vs. Ag/AgCl Potential (mV) vs. Ag/AgCl

Potential (mV) vs. Ag/AgCl Potential (mV) vs. Ag/AgCl
Fig. 7 Cyclic voltammograms with an electrolyte solution of 10 mM K3Fe(CN)6 in 1 M KNO3 for: (a) conventional Ti; (b) anodized Ti; and (c) MWCNT-Ti. (d) The capacitance of all the electrodes in comparison. (e) The plot of the square root of scan rates with anodic peak currents (I a)

and cathodic peak currents (I )

However, in Fig. 7a, b, the Ti and anodized Ti did not show any redox peaks. This is likely due to the inhibiting property of the titanium oxide on electron transfer. Figure 7a, b shows that bare Ti has less capacitance than MWCNT-Ti. The capacitance relationship is derived from: i = C(dv / dt), where dv /dt is a scan rate (Tamir et al. 2007). Hence, the capacitance between the working and reference electrodes during cyclic voltammetry was calculated by dividing the current in CV with respect to the specific scan rate, as shown in Figs. 7d and 9d. In summary, the electrochemical response of the MWCNT-Ti electrode promoted a higher charge transfer than the Ti and anodized Ti electrodes.

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