Biomimetics at the Nanoscale

#### **Chapter 5**

## Bio-Simulation of the Induction of Forced Resonance Mechanical Oscillations to Virus Particles by Non-Ionizing Electromagnetic Radiation: Prospects as an Anti-Virus Modality

*Nikolaos K. Uzunoglu*

#### **Abstract**

The induction of acoustic-mechanical oscillations to virus particles by illuminating them with microwave signals is analyzed theoretically. Assuming the virus particle is of spherical shape, its capsid consisting primarily of glycoproteins, a viscous fluid model is adopted while the outside medium of the sphere is taken to be the ideal fluid. The electrical charge distribution of virus particles is assumed to be spherically symmetric with a variation along the radius. The generated acoustic-mechanical oscillations are computed by solving a boundary value problem analytically, making use of Green's function approach. Resonance conditions to achieve maximum energy transfer from microwave radiation to acoustic oscillation to the particle are investigated. Estimation of the feasibility of the technique to compete with virus epidemics either for sterilization of spaces or for future therapeutic applications is examined briefly.

**Keywords:** virus mechanics, acoustic radiation biomedical effects

#### **1. Introduction**

The study of the physical properties of various types of viruses has attracted significant interest from several interdisciplinary research groups during the last 10 years [1]. Mechanical properties of virus particles with diameters of 10–300 nm, in particular the capsids enclosing the virus gene structures (DNA, RNA both single- and double-stranded), have been studied experimentally using atomic force and electron microscopy (AFM) [2, 3]. Also, elasticity theory methods are applied to conclude the mechanical properties of virus particles [4]. Electric charge distributions of the virus have also been studied by several researchers [5–9]. It is observed that under physiological conditions of salinity and acidity, virus capsid assembly requires the presence of genomic material that is oppositely charged to the core proteins [10]. Furthermore,

few researchers have focused their research on the possibility of inducing photonphonon interactions [11] in virions, which are the virus causing infections [12–17]. Already resonance phenomena of the H3N2 and H1N1 viruses have been demonstrated experimentally [18] , leading to a high rate extinction of them at a resonance microwave frequency near 8 GHz. The physical phenomenon attributed to this interaction is the separation of positive-negative electric charges on the body of the virus particles and the coupling of microwave energy through the interaction with the threedimensional bipolar electric charge distributions, generating mechanical oscillations at the same frequency. At specific microwave frequencies depending on the diameter and other properties of the particle [19, 20], primarily the dipole acoustic mode, have been claimed and strong coupling leading to high level virus killing rates have been demonstrated recently [19, 20]. The effects of hydration levels on the bandwidth of microwave resonant absorption induced by confined acoustic vibrations have also been studied [21]. It should be stated that the involved phenomenon is of non-thermal nature related to non-ionizing radiation, in this case being the I band microwaves (6– 10 GHz). Raman scattering phenomena [22] have also verified the existence of acoustic-mechanical resonance phenomena in virus particles [23].

The recent ongoing Covid-19 worldwide pandemic [24] and its severe consequences make it attractive to investigate the possibility of utilizing the abovementioned resonance phenomenon either in sterilization of spaces [25] such as clinics, public venues, hospitals or in the future as a therapeutic modality in some cases. In this direction, the possibility of utilizing similar methods used in microwave-induced hyperthermia, to raise the temperature of malignant tumors inside the human body, could be envisaged as a therapeutic modality. In fact, contrary to hyperthermia where usually lower frequencies of 27–2450 MHz are used [26], in this case much higher frequencies (6–8 GHz) were needed to be used. In some cases, ultrasound and laser radiation modalities have also been used, in clinical hyperthermia, along the lowfrequency microwave radiation using endo-cavitary radiators. However, in the present case, the interaction of non-ionizing radiation with tissues will be entirely different from hyperthermia to compete with the virus populations. The rather high frequencies used in the present resonance phenomenon pose a challenging problem to penetrate with high-intensity electric fields inside the human body such as in the case of the lungs. However, in the case of the larynx and throat and even some parts of the lungs, endo-cavitary radiators [27], as done in hyperthermia, could be used. Finally, since in the present case, the action of microwave radiation has the character of a resonance interaction, it is foreseen not to need longtime irradiation, contrary to hyperthermia, which in order to raise the tissues from 43 to 45<sup>o</sup> C needs usually 45–60 minutes. This principle allows to propose in present case short duration pulsedperiodic high-intensity microwave signals [28, 29]. This is expected to alleviate to some degree the penetration problem of electromagnetic energy to the human body.

In all the mentioned publications of this resonance phenomenon, the virus particle is assumed of being an elastic particle, as was modeled by H. Lamb in 1887 [30] for the oscillations of an ideal spherical isolated in space. In the present chapter, the mathematical analysis is carried out also considering the surrounding medium of the virus particle and taking into account the interaction of external microwave radiation with the electric charge distribution of the virus particle. Based on the recently published data on the structure [31] of the Covid-19 virion being 100–150 nm in diameter, because of its reach liposome capsid with few proteins on it, the present work leads us to adopt the model of the spherical virus particle as a viscous fluid while the outer space is taken to be an ideal fluid, with different acoustic characteristics of the

*Bio-Simulation of the Induction of Forced Resonance Mechanical Oscillations to Virus… DOI: http://dx.doi.org/10.5772/intechopen.106802*

spherical particle. Furthermore, vortex phenomena in modeling the viscous virus structure are neglected, since it is assumed that these are very weak and they have no effect on the resonance phenomenon to be studied.

The bio-simulation of the forced oscillations of a virus particle is carried out in the following mathematical steps presented with a flow diagram (**Figure 1**).

**Figure 1.**

*A flow chart diagram of the bio-simulation analysis of the interaction of virus particles with electromagnetic waves inducing acoustic oscillations inside the virus particle.*

#### **2. Mathematical formulation of the phenomenon**

A spherical particle of radius α, shown in **Figure 2**, is assumed to pose a continuous electric charge distribution with spherical symmetry defined by the equation

$$\rho\_q(\mathbf{r}) = \frac{Q}{\sigma} \left( \mathbf{1} - \frac{5}{3} \left( \frac{r}{a} \right)^2 \right) \tag{1}$$

where Q is the total positive electric charge in the center of the sphere, σ = 8πα<sup>3</sup> (3/5)3/2/15 is a normalization constant. The term 5/3 in the

Above Eq. (1) was selected to have the total charge of the particle to be zero, that is to have a balance between the positive (inner r<α(3/5)1/2 region) and negative (towards the external surface) charge distributions. It is evident that the particle could have the opposite charge distribution and the same analysis is valid. The proposed method is extendable to the case of non-symmetric charge distribution, and then higher order modes will be excited.

The spherical model of the virus is assumed to be a compressible fluid, characterized by its homogenous mass density ρ<sup>1</sup> acoustic wave propagation speed c1, and total viscosity constant (dynamic and bulk) χ. Then, assuming *ej<sup>ω</sup><sup>t</sup>* as time dependence, the propagation of acoustic wave phenomena is described by the following field equations [32]:

#### **3. Newton law**

$$
\rho\_1 j a \nu\_1(\mathbf{r}) = -\nabla P\_1(\mathbf{r}) + \mathbf{x} \nabla(\nabla \cdot \mathbf{v}\_1(\mathbf{r})) + \mathbf{f} \tag{2}
$$

where *v***<sup>1</sup>** is the velocity, *P*<sup>1</sup> the pressure field and *f* is the force density (N/m<sup>3</sup> ) because of the electric charge distribution inside the sphere.

**Figure 2.** *Spherical model for the virus particle.*

*Bio-Simulation of the Induction of Forced Resonance Mechanical Oscillations to Virus… DOI: http://dx.doi.org/10.5772/intechopen.106802*

#### **4. Mass continuity equation**

$$
gamma P\_1(\mathbf{r}) = -c\_1^2 \rho\_1 \nabla.v\_1(\mathbf{r})\tag{3}
$$

The force density term *f* in Eq. (2), taking into account the charge distribution given in Eq. (1) and the incident electric field *E*ð Þ¼ *r Eo*^*z* of the microwave radiation propagating along the x-axis and polarized parallel to the z-axis (see **Figure 2**), considering the size of the particle to be extremely small compared to microwave radiation wavelength, is obtained to be:

$$\mathbf{f} = \rho\_1(\mathbf{r}) E\_o \hat{\mathbf{z}} \tag{4}$$

Operating on the Eq. (2) the ∇*:* operator from the left-hand side, substituting Eq. (3), Eq. (4) and rearranging the terms the following wave equation is obtained:

$$
\nabla\_\cdot^2 P\_1 + k\_1^2 P\_1 = E\_o \mu\_o \mathbf{z} \tag{5}
$$

where

$$k\_1 = \frac{o\nu/c\_1}{\sqrt{1+j\varepsilon}}, = \frac{o\chi}{c\_1^2 \rho\_1}, \mu\_o = -\frac{10Q}{3\sigma a^2} \tag{6}$$

The pressure field outside of the particle assuming an ideal fluid is described by two respective equations of Eqs. (2) and (3):

$$
\delta j a \rho\_o \mathfrak{v}\_o(\mathbf{r}) = -\nabla P\_o(\mathbf{r}) \tag{7}
$$

$$
tau\_o(\mathbf{r}) = -c\_o^2 \rho\_o \nabla. \mathbf{v}\_o(\mathbf{r})\tag{8}$$

where *Po*ð Þ*r* is the pressure, *vo*ð Þ*r* the velocity, *ρο*the mass density and *co* the acoustic speed. Also combining Eqs. (7) and (8):

$$
\nabla^2 P\_o(\mathbf{r}) + k\_o^2 P\_o(\mathbf{r}) = \mathbf{0} \tag{9}
$$

where *ko* ¼ *ω=co* is the wave constant of the infinite space outside of the sphere.

#### **5. Solution of the boundary value problem**

#### **5.1 The field expression inside the sphere r<α**

The acoustic pressure inside the spherical particle being excited by the interaction of microwave electric field component acting to electric charges, being inside the spherical volume, could be described in terms of the primary (*P*10ð ÞÞ *r* and secondary (*P*11ð Þ*r* ) pressure fields [33].

In the analysis to follow spherical coordinates are used *r, θ* and *φ* being the radial distance from the origin, *θ* being the angle measured from z-axis and *φ* the azimuth angle.

The primary field *P*10ð Þ*r* should satisfy Eq. (4) with the right-hand side inhomogeneous term. Based on Green's theory, assuming the outside medium being infinite, the primary pressure is determined by using the equation:

$$P\_{10}(\mathbf{r}) = \mathfrak{u}\_o \iint\_{\text{Sphere}} \mathbf{G}\_1(\mathbf{r}, \mathbf{r}') \, \stackrel{\text{'}}{\text{z}} \, \stackrel{\text{'}}{\text{d}} \mathbf{r}' \tag{10}$$

inserting *z'=r'cos(θ')* and the expansion [34].

$$G\_1(r, r') = -jk\_1 \sum\_{n=0}^{\infty} j\_n(k\_1 r\_<) h\_n^{(2)}(k\_1 r\_>) \sum\_{m=-n}^{n} Y\_n^m(\theta, \phi) Y\_n^{\*m}(\theta', \phi') \tag{11}$$

where *j <sup>n</sup>*ð Þ*:* and *<sup>h</sup>*ð Þ<sup>2</sup> *<sup>n</sup>* ð Þ*:* are the spherical Bessel and Hankel (second type) functions,*r*<sup>&</sup>lt; ¼ min *r*, *r*<sup>0</sup> ð Þ and *r*<sup>&</sup>gt; ¼ max *r*, *r*<sup>0</sup> ð Þ, the angular spherical wave function

$$Y\_n^m(\theta,\,\varphi) = j^n \sqrt{\frac{2n+1}{4\pi} \frac{(n-m)!}{(n+m)!}} \mathcal{e}^{jm\rho} P\_n^m(\theta,\,\varphi) \tag{12}$$

and *Pm <sup>n</sup>* ð Þ *θ*, *φ* being the Legendre function*:*

Substituting Eq. (11) into Eq. (10) and *z'=r'cos(θ')*, the fact the double summation in Eq. (10) being limited on the terms m=0 and n=1 after the orthogonality of the angular wave functions and the Bessel functions integral [35]

$$\int\_{r=0}^{a} j\_1(k\_1 r\_<) h\_1^{(2)}(k\_1 r\_>) r'^3 dr' = a^4 j\_1(k\_1 r) w\_o + jr/k\_1^3$$
 
$$\text{with } w\_o = \left( 3h\_1^{(2)}(k\_1 a) - k\_1 a h\_0^{(2)}(k\_1 a) \right) / \left( a^2 k\_1^2 \right)$$

leads to the result of the primary pressure field

$$P\_{10}(r) = jk\_1 \mu\_o E\_o \cos\left(\theta\right) \left(a^4 j\_1(k\_1 r) w\_o + \frac{jr}{k\_1^3}\right) \tag{13}$$

Noticing that the primary field depends only to *Po* <sup>1</sup>ð Þ¼ *cosθ* cosð Þ*θ* angular function (n=1 and m=0 terms), the secondary pressure field is written easily:

$$P\_{11}(r) = A \, j\_1(k\_1 r) \cos \left(\theta \right) \tag{14}$$

#### **5.2 The field expression outside the sphere ( r>α)**

Considering the excitation of only the wave with cos(*θ*) dependence and the necessity of radiation condition to be valid for *r* ! þ∞ we can write easily:

$$P\_o(\mathbf{r}) = B \, h\_1^{(2)}(k\_1 r) \cos \left(\theta \right) \tag{15}$$

In Eqs. (14), (15), the unknown coefficients A and B are determined by imposing the validity of the boundary conditions at the spherical surface *r=α*:

Continuity of pressure fields *P*<sup>10</sup> þ *P*<sup>11</sup> ¼ *P*<sup>0</sup>

*Bio-Simulation of the Induction of Forced Resonance Mechanical Oscillations to Virus… DOI: http://dx.doi.org/10.5772/intechopen.106802*

Continuity of radial velocities ^*r:*ð*v***1**ð Þ� *r v***0**ð Þ*r* Þ ¼ 0

Then the A and B coefficients are calculated easily after some algebraic operations. The final solution for the secondary field inside the sphere and in particular for the total pressure is obtained to be:

$$P\_1 = a^{-2} Q E\_o \cos\left(\theta\right) W \tag{16}$$

$$W = -j2.569 k\_1 a \left[ \left( j\_1(k\_1 r) w\_o + \frac{jr}{a^4 k\_1^3} \right) + j\_1(k\_1 r) S \right]$$

$$S = T/R$$

$$T = \left( a k\_1 j\_1'(k\_1 a) w\_o + \frac{j}{a^3 k\_1^3} \right) h\_1^{(2)}(k\_o a) a^{-1} k\_o^{-1} - \left( j\_1(k\_1 a) w\_o + j a^{-3} k\_1^{-3} \right) h\_1^{(2)}(k\_o a)$$

$$R = j\_1(k\_1 a) h\_1'^{(2)}(k\_o a) - (1 + je) j\_1'(k\_1 a) h\_1^{(2)}(k\_o a) \frac{\rho\_o k\_1}{\rho\_1 k\_o}$$

#### **6. Numerical calculations**

After computing the pressure field as given in Eq. (16), it is shown that the "form factor" *W* is a function of the dimensionless quantities *k*1*a*,*koa*,ð Þ *ροk*<sup>1</sup> *=*ð*ρ*1*k*0), *r=α* and the parameter related to the viscosity of the virus particle *<sup>ε</sup>* <sup>¼</sup> *ωχ c*2 1*ρ*1 <sup>¼</sup> *koac*<sup>2</sup> *o c*2 1 � �*δ*, where *δ* ¼ *χ=*ð Þ *coρ*1*α* is a quantity related to the total viscosity of the spherical virus particle. Remembering that *<sup>χ</sup>* <sup>¼</sup> <sup>4</sup>*<sup>η</sup>* <sup>3</sup> þ *κ* [36], where *η*,*κ* are the shear and bulk viscosity coefficients of a Newtonian fluid, we take the quantity *δ* as a "measure of the degree of viscosity" in our calculations. Furthermore, the interest being on the maximum pressure on the particle, we take *θ* ¼ 0 or*π* on the two poles of the sphere where the rupture of the virus capsid is sought.

In **Figure 3**, numerical results of the *W* "form factor" are given in the range 0*:*1<*koa*< 3*:*0 for various parameters of the ratios *ρο ρ*1 ¼ 1*:*05,1*:*1,1*:*2 and 1*:*3, c0=1560m/s (speed of sound in the outer space), c1 = 1950 m/s (speed of sound inside the sphere) . Meanwhile, the viscosity parameter is taken χ = 0.01 and χ = 0.001, which corresponds to the total viscosity constant corresponding to δ = 0.1 and δ = 0.01 (N∙*s=m*<sup>2</sup>Þ. The numerical results show interesting resonance behavior when viscosity is *δ* = 0.01. The phenomenon is stronger as *δ* decreases.

It is well known that the scattering of incident waves to a sphere (acoustic or electromagnetic waves) shows resonance phenomena when the refractive index of the spherical scatter has a large value. This phenomenon is well known in classical and quantum physics (Regge poles). As mentioned in the introduction section several researchers have foreseen this phenomenon. However, in the present analysis, the adopted model and analysis takes into account, although in a simplified form, all the involved mechanisms. The resonance is occurring near the angular frequency *ω=πco/(2a)*, which corresponds to the "dipole mode" of the spherical particle.

In order to assess the feasibility of utilizing the phenomenon to compete with the virus populations, we need to calculate the pressure being developed at the spherical surface. Placing in Eq. (16) *r = α*, 2*α* = 100 nm and *Q = Neo, eo*=1.62 x 10�<sup>19</sup> Cb (electron charge), N being the number of + or – charges we obtain after Eq. (16) the

#### **Figure 3.**

*Dependence of W (Eq. (16)) function to* koα. *The W function is the "form factor" of the resonating spherical virus particle and the resonance takes place when the R term takes maximum value at a specific microwave frequency, which is inducing mechanical-acoustic oscillation through electric force interaction between electromagnetic wave and electric charges.*

pressure *P1* =4∙10�<sup>5</sup> ∙*N Eo W,* since *W* � 2*:*000 see ð **Figure 3**) following the data given in ref. [9], the surface electric charge being *σ* ≤0*:*5*eo=nm*<sup>2</sup> the virus area being *As = 4πα* 2 , we obtain *<sup>N</sup>* � <sup>3</sup>∙10<sup>4</sup> and *<sup>P</sup>*<sup>1</sup> �*2.400 Eo* (Pa)*.* Then, if the imposed electric field at microwave frequency is Eo = 1.000 (V/m) (this corresponds to a power density of 130 mW/cm<sup>2</sup> much less used in hyperthermia treatments many times being 15.000 mW/cm<sup>2</sup> as a continuous wave signal), we arrive to the estimation that the pressure oscillation amplitude exerted on the two poles of the virus will be *P*<sup>1</sup> � 2*:*4 MPa. The

**Figure 4.**

*Dependence of W [Eq. (16)] function to koα in case of absence of viscosity.*

*Bio-Simulation of the Induction of Forced Resonance Mechanical Oscillations to Virus… DOI: http://dx.doi.org/10.5772/intechopen.106802*

mentioned microwave field-generated pressure wave on the capsid surface seems to be comparable with the bulk Young's module being equal to 5 MPa (see the end of the section 'Methods' of ref. [8]).

In **Figure 4**, computations in the case of zero viscosity are presented for various ratios *ρ*1*=ρο*. The strong resonance phenomenon of the ratio *ρ*1*=ρο* is 1.3 while the lowering of the resonance frequency as this ratio decreases while the peak value of *W* has some variation.

The above initial results show that the argument expressed by the National Taiwan University in their seminal paper of ref. [18] is verified theoretically with the present model.

The microwave resonance frequency is computed easily known the value of *k\*= koa* the peak value is attained, that is *fresonance (Hz)=k\*c/(2πα)* where c=3.10<sup>8</sup> (m/s) is the speed of electromagnetic waves in vacuum.

#### **7. Conclusions**

A simplified model of a virus particle allowed to analyze the coupling phenomena between microwave (electromagnetic) radiation and acoustic waves generated inside the particle. Based on recent publications on virus physical and electronic properties of viruses, similar to Covid-19, computations show the possibility of strong interactions to generate rupture or capsid of the viruses. This action is based on the Coulomb force exerted by the oscillating field on the inhomogeneous electric charges within the spherical particle. The microwave resonance frequency—which is identical to the acoustic wave—is in the region of 6–10 GHz.

The prospect of using the presented principle to sterilize public spaces, hospitals, clinics etc. is an attractive proposition. The present microwave technology is available for the development of this type of portable device. Moreover, the existing more than 40 years of experience in clinical hyperthermia, which is based on the use of low microwave frequencies as an adjuvant therapy to treat many cancer diseases, makes it attractive to investigate the possibility of developing technologies to implement the mentioned idea in the future to depopulate virus populations inside the human body. Before this, extensive in vitro trials in virus and cell cultures need to be carried out to follow with animal trials as well.

#### **Author details**

Nikolaos K. Uzunoglu National Technical University of Athens, Athens, Greece

\*Address all correspondence to: nuzu@central.ntua.gr

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 6**

## Assessment of the Addition of Fluorapatite in Hydroxyapatite Coatings: Implementation Prosthetics/Bone in Vivo

*Halima Feki Ghorbel, Awatef Guidara, Yoan Danlos, Jamel Bouaziz and Christian Coddet*

#### **Abstract**

Hydroxyapatite (Hap: Ca10 (PO4)6 OH2)-Fluorapatite (Fap: Ca10 (PO4)6F2) composite coating on 316 L stainless steel, using the High-Velocity Oxy-Fuel Spray (SHVOF), was investigated. This work is an evaluation of the bioactivity of bone/ Hap–Fap composite coatings implanted in the tibia of the rabbit. A small amount of Fap (6.68, 13.26 and 26.52 w% Fap attributed to 0.25, 0.5 and 1% fluor) was introduced into Hap. The fluorapatite provides more stable and adherent deposits. The characteristics of the coatings were investigated with various instruments including Scanning Electron Microscopy (SEM) and X-Ray Diffraction (XRD) and biological (in-vivo and in-vitro) tests. Hap−Fap coating showed excellent behavior in vitro and in vivo tests revealing that the Fap is effective in improving biocompatibility and bioactivity. This study draws inspiration from technological and biological selection solutions adopted by evolution, to transpose the principles and processes of human engineering.

**Keywords:** Fluorapatite, hydroxyapatite, high-velocity oxy-fuel spray, In-vitro/In-vivo test

#### **1. Introduction**

Research has never ceased trying to improve materials for bone and dental implants and the techniques used to synthesize them. To begin with, metals were the most widely adopted materials for implants. However, they were not without problems. Indeed, Schulze et al. assessed the effect of In-Vivo exposure to metallic nanoparticles on bone marrow In-Vitro, revealing significant alterations in cell biology [1]. Similarly, in Total hip arthroplasty 'THA', the problem of implant loosening due to aseptic osteolysis, was observed to be triggered by wear particles from the implant articulating surfaces. The overall In-Vivo performance of metallic hip implants turned out to be comparably poor and caused some manufacturers to recall their products. Thus, several researchers turned to the study of various bioactive

ceramics, such as Hydroxyapatite (Hap), Tricalcium phosphate (TCP) and Bioglass, developed to activate bone regeneration [2]. Given its similar chemical and physical characteristics to bone, Hap was widely used as bone substitute material in orthopedic domain [3, 4]. However, when dispersed as nanoparticles Hap can cause inflammation by activating monocytes and neutrophils [4–6].

Within this prospect, Fluorapatite (Fap) was initially chosen for this purpose because of its fairly good biological and mechanical properties compared to β-TCP and hydroxyapatite (Hap) when used alone. Nevertheless, the long-term stability of bioactive ceramic implants was criticized for at least two shortcomings: the presence of low solubility of the coating and the absence of high adhesion strength between coating and substrate [7]. Hence, as a solution to these problems, Dhert and Cheng proposed to maintain F-concentration down to the minimum level in order to decrease the solubility of fluorohydroxyapatite (FHa) [8, 9]. In a similar attempt to decrease the solubility of the coating, Wolke et al. recommended the use of a fluoride coating containing Hap [10]. Indeed, Hap and Fap constitute the inorganic compound of the human hard tissue. Unfortunately, Baltag et al. and Overgaard et al. reported that the high degradation rate of Hap coating in biological environments is a serious concern, which might be harmful to adhesion properties, resulting in undesirable debris and even delaminating, which eventually leads to the failure of the implant [11, 12]. Furthermore, the stability of Hap−Fap composites was also a source of concern because dissolution or reprecipitation influences cell behavior [13]. Indeed, when Hap−Fap composites are suspended in SBF liquid, they can dissolve and precipitate rapidly, leading to ion release, change in the pH, size, and morphology [14, 15].

As a solution to these problems, some studies [16, 17], presented Fap as potential replacement for Hap in implants because of the higher degradation of the latter material in biological environments and its lower adherence. For these reasons, other researchers worked on developing the mechanical properties of coatings from Fap and Hap together to increase their efficiency. Bahandag et al. [17] studied the influence of Fap on the properties of Hap coating using thermal projection. These scholars revealed that Fap increases Hap coating crystallinity. Furthermore, the slow release of Fap reduces the delivering particles leading to the improvement of its osteointegration. Similarly, Chang et al. [18] showed that the fluorine ions improve the osteoblastic cells' proliferation and differentiation. Hence, scholars such as Fraz and Telle and Somrani opted for Fap as an additive to Hap and a replacement for pure Hap coating on metallic implants because of their chemical composition which is similar to the bone mineral [19, 20]. Mark and Brown confirmed this finding and explained that these materials presented good biocompatibility [21]. Particularly in dental prostheses, fluor is effective in inhibiting caries [22] and proves to be compatible with the human bone that contains approximately 1 wt% of fluor [23–25].

The second issue related to implants was the technique of synthesizing the chosen materials. Thermally sprayed bio-ceramic on metallic substrate was widely used in orthopedic prostheses given its great potential in bone regeneration activity In-Vivo. Among the adopted Ceramic coating techniques are plasma spraying, flame spraying and high-velocity oxy-fuel (HVOF) spray [26]. The Plasma spray process is the most commercially preferred technique for clinical applications. The use of bond coats to improve the surface properties of metallic substrates was also studied extensively in the thermal spray literature [27]. However, despite the abundance of research on Hap coating and on the mechanical properties and bioactivity of Fap, there is still a serious need to improve the synthesis, characterization and application of Fap−Hap coating composite. For this reason, this work intended to investigate the Fap (6.68; 13.26 and

*Assessment of the Addition of Fluorapatite in Hydroxyapatite Coatings: Implementation… DOI: http://dx.doi.org/10.5772/intechopen.107376*

26.52 w%)-Hap coating on the 316 L stainless steel using the SHVOF technique. The remainder of this paper is divided into three sections. Section two will present the characterization of raw materials and of as-sprayed apatite coating. Section three will exhibit the study of the bioactive behavior of samples using the simulated body fluid (SBF). Finally, section four will discuss the adhesion and biocompatibility of Hap and Hap-Fap coating on rabbit bone cells as applied through the SHVOF technique.

#### **2. Materials and methods**

#### **2.1 Materials**

The Fap powder was synthesized using a wet-chemical method [20]. A calcium nitrate solution was slowly poured into a boiling solution containing di-ammonium hydrogen-phosphate, and a 28% NH4OH solution was added to the mixture in order to adjust the pH to 9. The precipitate was filtered, washed, dried at 70°C for 12 h and calcinated at 500°C. The Hap powder with Ca/P ratio of 1.66−1.71 (Medicoat, HA-15-183, 95%) was used as base material. The Hap−Fap powders were mixed using a dried-mechanical method.

#### **2.2 Methods**

The Hap and Hap−Fap coatings were carried out on 316 L stainless steel discs by High-velocity oxy-fuel spray (SHVOF) using a Sulzer-Metco F4-MB (Switzerland). Before applying the coating, degreasing and grit blasting were carried out to make the substrate surface coarse and clean. The thermal sprayed process parameters are listed in **Table 1**. The coating thickness was evaluated using a micrometer (200 μm).

The simulated body fluid (SBF) which has ionic concentrations very similar to those of human plasma was used to study the bioactive behavior of samples. For this, a commonly used SBF solution of pH 7.4 was prepared according to the procedure recently described by Kokubo et al. [28]. **Table 2** presents the ionic composition of the as-prepared SBF and compares it with that of human plasma. The Hap and Hap− Fap coatings were cut in parallelepipedic slices of size 2\*6\*12 mm and then cleaned before being immersed in 100 ml of SBF. The temperature was maintained at 37°C.

The phase compositions of the coated samples were examined with an X-ray diffractometer (PHILIPS PANALYTICAL) and with a scanning electron microscope (JEOL JSM 5800LV). The roughness of the surfaces of both substrate and coatings was measured using Mitutoyo (ISO 1997).

Prosthesis implantation, for an "In Vivo" study in rabbits, was carried out in a laboratory of animal experimentation and approved by the faculty of medicine of Sfax and by the ethical committee of the Habib-Bourguiba University Hospital, Sfax;


**Table 1.** *Spray process parameters.*


#### **Table 2.**

*Ion concentrations in supersaturated SBF solution prepared in the present study and in human blood plasma.*

Tunisia. The surgical operation was performed on the rabbit, by an appropriate medical team with respect for all the rules of asepsis. The shape and dimensions of prostheses are shown in **Figure 1**. They were carefully chosen after several graft tests in order to perfectly fill in the medullar canal of the Rabbit tibia.

These prostheses were then sterilized by 60CO gamma irradiation (Equinox, UK).

Five adult white rabbits, aged from 5 to 10 months, were used for the experiments. All surgical procedures were done under strict aseptic protocol. The animals were premedicated and anesthetized with a xylasine/ketamine mixture (10 mg/kg). The animal tibialis anterior face was shaved. After the injection, the animal was left at rest with its eyes closed for about 20 minutes. The skin was disinfected with a povidoneiodine solution of 10% (Betadine, Medapharma). Local anesthesia (Unicaine 2%) was employed in the anterior tibialis face. Four cylindrical bars with a length of 37 mm and diameter of 4–5 mm (**Figure 1**) were placed in each animal. Surgical preparations for the cylinders were done using first a pilot drill and then a 2 mm twist drill. Careful drilling was done with a low rotary handpiece. The skin was sutured with interrupted

#### **Figure 1.**

*Prostheses dimensions for implantation: Great diameter: 5 mm; small diameter: 4 mm; degree of inclination: 30° and length: 37 mm.*

*Assessment of the Addition of Fluorapatite in Hydroxyapatite Coatings: Implementation… DOI: http://dx.doi.org/10.5772/intechopen.107376*

threaded sutures. Skin was again soaked with a povidone solution. After 28 days the rabbits were sacrificed, and the implants extracted. The samples were stored in a formalin-based solution called BB'S and then included in methacrylic resin to be used for radiological testing of bone tissue. X-ray radiography was performed using a Faxitron X-ray system (Edimex, Angers, France) equipped with a camera (5X5 CCD). The coatings were characterized by Scanning Electron Microscopy (SEM) and X-Ray Diffraction (XRD). Moreover, the bonding strength of the as-sprayed coatings was measured using a universal testing system. The microstructure of the detached surfaces was examined using SEM.

#### **3. Results and discussion**

#### **3.1 Characterization of the different powders**

**Figure 2** shows the XRD patterns for Hap and Hap-synthesized Fap composite. This observation can have four implications. Firstly, Hap-diffraction peaks shown in **Figure 2a** can be indexed as typical hexagonal phases (ICDD 77−0120). These peaks situated at approximately 25.9, 32, 33.1, 40.1 and 46.7 correspond well to (002), (211), (300), (212) and (222) lattice planes of the classic hexagonal phase Hap-diffraction peak (a) can be indexed as typical hexagonal phase (ICDD 77−0120). Secondly, it was clearly observed that the addition of Fap in Hap did not affect the diffraction peaks location shown in **Figure 2b**–**d**. However, the increase of Fap quantity in Hap powders shown in **Figure 2b**–**d** slightly increased the diffraction peaks intensity. This observation, in total agreement with the standard requirements, indicates that the crystallinity was well preserved. Thirdly, this observation confirms Feki-Ghorbel et al.'s previous claim that the crystalline stability of Fap is greater than that of Hap [29]. Finally, this observation shows that there were no impurity diffraction peaks or phases in the XRD patterns of the Hap and Hap−Fap.

#### **Figure 2.**

*XRD patterns of powders: (a) hap; (b) hap−Fap (0.25 w% F); (c) hap−Fap (0.5 w% F) and (d) hap−Fap (1 w% F).*

#### **3.2 Characterization of as-sprayed apatite coating**

#### *3.2.1 SEM analysis*

The SEM micrograph shown in **Figure 3** reveals a characteristic lamellar microstructure of thermal spray coatings. A typical surface microstructure HVOF sprayed Hap coatings are generally porous. The porous structure might be beneficial to the

#### *Assessment of the Addition of Fluorapatite in Hydroxyapatite Coatings: Implementation… DOI: http://dx.doi.org/10.5772/intechopen.107376*

biomedical application involving the mechanical fixation by bone in growth. Firstly, indicate where you can see the microstructures in terms of **Figure 3a**–**c**.

Secondly, these observations were not the result of the same quantity of Fap. Therefore, the differences in microstructures can be the result of the quantity of Fap. This should be clearly explained. The microstructure of the Hap and Hap−Fap coatings consists of particles of different shapes and sizes: fully molten splats which are merged in each other (white circles in the photos), some small globular particles in the matrix give a dense appearance and others probably are the un-melted particles. No crack can be observed.

#### *3.2.2 XRD analysis*

**Figure 4** shows the XRD patterns for Hap and Hap−Fap as-sprayed coating. We can observe the presence of the pattern peaks attributed to Fap and Hap. Diffraction peaks of Hap−Fap coating showed more intensity than that of Hap coating. This can be interpreted as an indication that the addition of Fap increases the crystalline phase of Hap coating. Hence, our XRD analysis confirms once more Feki-Ghorbel et al.'s previous claim that the crystalline stability of Fap is greater than that of Hap [29]. In addition, this result suggests that the HVOF spray parameters were optimized. Finally, the observation of a little amorphous phase implies that Fap can be chosen as a potential partial substitute for Hap.

#### *3.2.3 Surface roughness*

The surface properties of implants are of vital importance for implant tissue interaction which further influences the biocompatibility for clinical use [30, 31]. Particularly, surface roughness alters osteoblastic attachment proliferation of bone cells and their differentiation and matrix production [32, 33]. The surface roughness parameters (Ra, Rq and Rz) for blasted stainless steels and for 316 L substrates coated by Hap and Hap− Fap are shown in **Figure 5**. The HVOF spray treatment clearly modified the surface roughness of the samples (Ra). In fact, the average surface roughness of blasted steel substrate increased from1.5 to 2.9 ± 0.2 μm. Moreover, the addition of Fap improved the

**Figure 4.** *XRD patterns of powders: (a) hap; (b) hap−Fap (0.25 w% F); (c) hap−Fap (1 w%F).*

#### **Figure 5.**

*Surface roughness measurements for the SHVOF sprayed hap; hap-Fap-coated 316 L along with that of the grit blasted uncoated 316.*

average surface roughness which reached 4.7 ± 0.2 μm for the Hap−Fap (w% 1 fluor). Such results are relevant for the clinical application of Hap−Fap coated implants. As was reported by authors [34, 35], the modification of the surface roughness of an implant significantly influences In-Vitro osteoblastic response. Furthermore, a better long-term In-Vivo response of the implant is achieved when the surface roughness is increased as the amount of bone in direct contact with the implant surface as well as the loads and torques required for extracting the implant from bone growth increase [28].

The comparison between the surface roughness results obtained in this work and those of the reported HVOF-sprayed and the flame-sprayed Hap coating [36–38] shows that the Hap−Fap coatings presented a comparative surface roughness while the addition of Fap yielded a higher roughness of the surface.

#### **3.3 In-vitro tests**

#### *3.3.1 XRD analysis*

**Figure 6** presents the XRD patterns of SHVOF Hap−Fap (0.25 and 1 w% F) composite coatings in the function of immersion time in SBF. After each immersion time in SBF (0, 3 and 28 days), the examination of the coating surface shows the expansion of the diffraction lines group around 37° (2θ) corresponding to Hap phase. After a 28-day immersion in SBF, the XRD pattern presented a diffraction halo situated between 35° and 40° (2θ). The mineralogical phase analysis, using the software "High score", allowed identifying the transition phases appearing on the Hap−Fap surface. The diffraction patterns can be attributed to a hydrated carbonated apatite layer. These patterns included lines of diffraction which can be mainly attributed to a carbonated apatite [00–012-0529]. An intensity peak around 34.5° which can be attributed to a.

Hydrated Apatite [01–077-0128] was also detected. The crystalline structure modification after a 28-day immersion in SBF produced a formation of a new carbonated apatite precipitate covering all the Hap−Fap (0.25 w% and 1 w% F) surface.

*Assessment of the Addition of Fluorapatite in Hydroxyapatite Coatings: Implementation… DOI: http://dx.doi.org/10.5772/intechopen.107376*

**Figure 6.**

*XRD patterns of SHVOF hap−Fap composite coating in function immersion time in SBF: 0.25 w% F (a) and 1w% F (b).*

#### *3.3.2 SEM morphologies*

The bioactivity and biocompatibility of the Hap coating and Hap−Fap composite coatings were evaluated by immersing the samples in SBF for 0; 3; 7 and 8 days.

As shown in **Figure 7**, spherical-shaped particles were observed on the Hap surface of the coated material after three days of immersion in SBF. **Figures 8** and **9** confirmed with certainty the appearance of the new crystals on the Fap phase of the Hap−Fap composite on Hap−Fap at 0.25 and 1w% composite. As the immersion time increased, the crystals seemed to grow in size and to form an Apatite layer as shown after A 28-day immersion in SBF. This layer appeared to consist of many nano-sized crystallites with spherical morphology, as was observed by previous authors [39]. Feki-Ghorbel et al. [29] revealed that the incorporation of 1 w% of Fap into Al2O3 promoted the apatite layer formation on the coating surface when soaked in SBF. According to Kokubo [28], the bone-bonding ability of a material depends on the ability of apatite to form on its surface in SBF. In light of this idea, we can say with much certainty that since we observed the appearance of the apatite crystals on the Fap phase of the Hap−Fap composite surface, then we expect to reach very encouraging results in the in vivo tests.

**Figure 10** presents the micrographs of the SHVOF coating/bone interface following sacrifice of the animal 28 days after the implantation.

The Hap (**Figure 10a**) and Hap−Fap (**Figure 10b,c**) composite coating exhibits a good adhesion with bone. The addition of Fap encourages the presence of crystalline forms interconnected and constituting an array at the Hap−Fap/Bone interface.

A Cross-section observation also confirms the presence of a thin apatite layer on the Hap-composite surface as shown by In-vitro tests. In this work, we have observed that the incorporation of a little amount of Fap (1w%) into Hap promoted the apatite layer formation on the coating surface and could achieve bone-bonding when implanted in bone tissues, as do bioactive ceramics.

The understanding of the biological mechanisms involved in osteoconduction seems complex. In fact, the correlation between the microstructure and the biological activity

**Figure 7.** *SEM observation of hap surface after immersion in SBF solution.*

can intervene to be able to interpret the results. Nevertheless, two facts should be kept in mind when dealing with calcium phosphate films. Firstly, as was previously stated [40, 41], the bioactivity of the calcium phosphates coating depends on the capacity of their surface nucleated crystallized carbonated apatite, like the osseous mineral from the biological fluids. Secondly, the calcium phosphate films are crystallized under

*Assessment of the Addition of Fluorapatite in Hydroxyapatite Coatings: Implementation… DOI: http://dx.doi.org/10.5772/intechopen.107376*

#### **Figure 8.**

*SEM observation of hap−Fap (0.25 w% F) surface after immersion in SBF solution.*

conditions such as the formation of the random nucleated spherulites, which can be considered as only a stage essential to the mechanism of the osteo-conduction. Hence, it can be concluded that the structural composition of the biomaterial's surface has an influence on bioactivity. In this study, the addition of the various percentages of Fap in the Hap coating acted on the surface's physical state and so increased its bioactivity with nucleated apatite spherulites on the biomaterial surface. As regards Hap/Fap composite

**Figure 9.** *SEM observation of hap−Fap (1w% F) surface after immersion in SBF solution.*

coatings, after 28 days of immersion in the SBF and in contents raised in Fap (1% F), the observed transition phase can prove the presence of an amorphous neo-formed structure on the surface justifying the samples bioactivity.

Indeed, the important solubility of the amorphous phase increases the SBF oversaturation when in contact with the sample surface. Therefore, it triggers the nucleation of a neo-formed film similar to the osseous mineral. The SEM study of the *Assessment of the Addition of Fluorapatite in Hydroxyapatite Coatings: Implementation… DOI: http://dx.doi.org/10.5772/intechopen.107376*

#### **Figure 10.**

*SEM observation of a. hap/bone interface (a); hap−Fap (0.25w%F)/bone interface (b) and hap-Fap (1 w% F) / bone interface (c).*

surface film morphology after 3, 7 and 28 days of incubation showed a large amount of extra-cellular matrix proliferation.

This matrix stems from the crystallized apatite layer which triggered the osseous regrowth. It is important to note that this new apatite phase will result in osteoconduction. The physicochemical reactions occurring on the surface of bioactive materials could be decomposed into several stages described in the in vitro analysis above. Thus, the development of a layer of amorphous calcium phosphate occurred after a stage consisting of relegated ions forming a network of modifiers present in the deposits of the Hap−Fap matrix.

In chemical terms, this implies that Ca2 + ions present on the bone surface are quickly exchanged with the ions H+ present in the SBF. Hence, the amorphous precipitates of phosphate of calcium grow on the surface. In addition, the migration of the ions Ca2 + on the bone surface, the presence of PO4 3− in the Hap−Fap matrix and the existence of soluble phosphates in the SBF promote the bioactive phase formation.

At this stage, the amorphous calcium phosphate layer covers the coating surface containing an important amount of bioactive Fap. The calcium phosphates are crystallized thanks to the presence of the ions OH-, Mg2+ and carbonates CO3 2− in the middle. Then, the apatite carbonated phase occurs close to the bone mineral phase.

A Previous study, [16, 17] presented Fap as potential replacement for Hap in implants because of the higher degradation of the latter material in biological environments and its lower adherence.

#### **4. Conclusions**

To conclude, this investigation resulted in four important findings.

Firstly, this study demonstrated that the addition of Fap would produce a higher roughness at the bone surface which would certainly enable the osteoblastic cells to grow more easily. This would be very likely due to the fact that the more the surface of the bone is rough, the more it provides direct contact with the implant surface and the more it requires loads and torques for extracting the implant.

Secondly, the SEM observation of In Vitro tests confirms the formation of an Apatite layer on Hap and Hap−Fap coating after a 28-day immersion in SBF. The addition of 0.5w% and 1 w% Fap in the Hap coating increases the bone surface bioactivity through the growth of nucleated apatite spherulites on the biomaterial surface. In addition, it reduces the Hap phase solubility and leads to a stable amorphous layer.

Thirdly, this study revealed that the exchange of the calcium ions " Ca2 + " and the phosphate ions " PO43− " between the Hap-Fap matrix and the SBF promotes the bioactive apatite phase formation. This new apatite phase leads to osteo-conduction.

Fourthly, In Vivo tests showed partial resorption of the implant in the form of zones less dense than the bone. The presence of this area would indicate the new bone growth accompanied by a slight osteointegration.

Our results suggest that the addition of Fap into Hap is more suitable for implantation of prostheses and for the study of bone substitutes. We can conclude with certainty that it represents the solution of choice the principles and processes of human engineering.

The bioactive behavior comparison of the two composites Hap-Fap with 0.25 wt% F and 1 wt% F revealed that the increased Fap level until 1 wt% F is presented as a better choice because it allowed the implant to develop a better integration which will guarantee a more durable life.

#### **Acknowledgements**

We would like to thank the medical team at the University hospital of Habib Bourguiba, Sfax, Tunisia for their valuable help in conducting the in vivo implantation on the rabbit in their laboratory.

*Assessment of the Addition of Fluorapatite in Hydroxyapatite Coatings: Implementation… DOI: http://dx.doi.org/10.5772/intechopen.107376*

#### **Author details**

Halima Feki Ghorbel1,2\*, Awatef Guidara1 , Yoan Danlos2 , Jamel Bouaziz1 and Christian Coddet2

1 LCI, Ecole Nationale d'Ingénieurs de Sfax"ENIS", Tunisia

2 LERMPS, Université de Technologie de Belfort-Montbéliard, France

\*Address all correspondence to: ghorbel.halima@gmail.com; halima.ghorbel-feki@uvsq.fr

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 4
