**1. Introduction**

Design requirements imposed on contemporary structural members frequently make it necessary to use materials that combine different strengths and mechanical properties. Examples of such materials include composites, particularly laminates, which may consist of several layers of materials with different strengths and mechanical properties, selected depending on the specific needs of the user. Usually, one of the layers of laminate is responsible for structural strength, while the remaining layers may have special properties—corrosion protection, thermal insulation and sealing or damping of high-energy impact loads. The object under test described in this paper is an Al–Ti laminate consisting of three layers: outer lamella from an Al alloy (AA2519), inner lamella from an Al alloy (AA1050) and an outer lamella from a Ti alloy (Ti6Al4V).

Various techniques and technologies are used during production to join different metallic materials in the laminates—adhesive bonding, high-temperature welding, friction welding [1–4] and, increasingly, explosion welding [5–9]. This technology uses the explosive energy for mutual penetration of the bonded materials. The technology of explosion bonding is fairly new, and it is used to make various types of items consisting of different materials; in each specific case, the parameters of the process are selected based on experience with laboratory specimens. That is why, in order to use elements made of layered materials created by explosion welding, it is necessary to know their strength and mechanical properties. The least investigated and, at the same time, very important area, which frequently determines the strength of the layered material, is the interface of the bonded materials—lamellas. There are no theoretical

studies concerning explosion bonding of different materials and mechanical characteristics of the created laminates, as a result of which the most reliable method is to assess their strength properties using experimental means [10–15]. On the other hand, the commonly applied methods of determining material characteristics have been prepared for homogeneous materials, which is why it is difficult to apply them directly to laminates, and, consequently, the determined physical and mechanical properties cannot be regarded as the actual material characteristics of the laminates.

The AE technique is commonly used to detect and monitor damage and development of such damage in various structures, and it is currently recognised as one of the most reliable and well-established methods of non-destructive testing (NDT) [16]. Acoustic emission is a very e fficient and e ffective method of detecting cracking and fatigue of metals, glass fibre, wood, composite materials, ceramics, concrete and plastics [17–19]. It can also be used to detect faults and pressure leaks in tanks or pipes or monitor the progress of corrosion in welded joints [20].

Unlike other techniques, which can only detect geometric discontinuities, AE methods can detect fibre tearing, delamination of adjacent layers in laminated composite plates, matrix cracking and fibre pull-out [21]. Most AE signals are caused by friction or by friction among the damaged components of the composite. Potential application of the AE technique to the assessment of damage to composite materials was discussed in References [22,23], with the mechanisms behind the cracking of composite materials being described in Reference [23]. Studies of local damage to composite materials based on an analysis of the acoustic emission signal were carried out by Marec et al. [24]. The results of the tensile tests clearly identified damage mechanisms in various composite materials: cross-ply composites and sheet moulding compound (SMC). The tests indicated an evolution of damage in these materials over time until global failure, and they identified the most critical damage mechanisms. It was also found that the generated AE signals were subject to scattering and attenuation due to the elasticity of the waves. Most studies carried out in order to monitor AE signals in metals were focused on samples with the shape of thin plates [16,25]. However, the propagation of waves in thin plates is dispersive by definition [26] due to the different phase velocities at which different frequencies are propagated. Dang Hoang et al. [27] showed a relationship between the duration and energy of the AE signal and failures in aluminium plates joined together. In a similar study, the relationship between AE count rates and crack propagation rates in welded steel samples during fatigue was described in Reference [28]. Aggelis et al. [17] established that certain parameters of AE signals, such as the rise angle (RA), duration and rise time, are very sensitive to crack propagation rate, and can be useful in the characterisation of damage if the AE signals from rise time (RT) samples subject to fatigue are accordingly interpreted. The tests described in Reference [29] regarding the fatigue properties of steel and welds and fractographic and microstructural observations show that AE can be used as a tool to monitor damage caused by fatigue of the structure due to the fact of its sensitivity to changes within the crack.

That is why using AE in the study made it possible to identify certain peculiarities of the fracture process in the tested layered composite. Also, the AE signals were clustered depending on the processes that generated them using the iterative *k*-means method, which clusters AE parameters in a Euclidean space [30,31], and analysed them using waveform time domain, waveform time domain (autocorrelation), fast Fourier transform (FFT Real) and waveform continuous wavelet based on the Morlet wavelet [32].

Understanding the processes occurring in the contact areas of di fferent materials has an important role in assessing laminate strength. Actually, these areas of contact of di fferent layers are the most important in composites strength analysis. Theoretical and experimental studies on the e ffect on bonding strength are presented in References [33–35].

This paper presents the results of studies of the fracture process in Al–Ti laminate. To implement di fferent fracture mechanisms the tests were carried out at two temperatures (i.e., *T*1 = 20 ◦C and *T*2 = −50 ◦C) on specimens with single-edge notches loaded in three-point bending (SENB). The force, specimens' deflection, crack opening and acoustic emission (AE) signals were recorded during the loading. Based on mechanical sensors signals the fracture toughness characteristics were obtained and di fferences in loading diagrams were established. Observation of the specimens' fracture surfaces

using scanning electron microscope (SEM) showed some peculiarities of the cracking process of Al–Ti laminate in tested temperatures. Great attention was given to the analysis of AE signals to identify fracture mechanisms of Al–Ti laminate.

### **2. Material and Testing Methods**

The object subject to laboratory tests was an Al–Ti laminate consisting of three layers: outer lamella from an Al alloy (AA2519), with a thickness of approximately 4.6 mm, inner lamella from an Al alloy (AA1050) with a thickness of approximately 0.2 mm, and an outer lamella from a Ti alloy (Ti6Al4V) with a thickness of approximately 4.6 mm. After the individual lamellas were bonded by explosion welding, the laminate was subject to heat treatment by soaking at 550 ◦C for 2 h, cooling to 165 ◦C and soaking at that temperature for 10 h. The authors of this paper analysed the fracture process in the laminate using specimens that had already been subject to welding and heat treatment. Information about the standard properties of base materials, welding process and heat treatment of the examined laminate was taken from References [12,15].

Experimental tests were carried out by the research team using SENB specimens (B = 10; W = 20; S = 80 mm) in three-point bending with a single-edge notch passing across all layers to a depth of 0.5W (Figure 1) at two temperatures: *T*1 = 20 ◦C and *T*2 = −50 ◦C. The strength characteristics of the component materials alloy at di fferent temperature are given in Table 1. The specimens were prepared and loaded in accordance with ASTM E1820-09 [36]. During loading, the specimens were partially unloaded in order to determine the change in the compliance and calculate the growth of the crack. Tests at lowered temperature *T*2 = −50 ◦C were conducted in a thermal chamber in the environment of nitrogen vapours. During the tests, temperature variations did not exceed Δ *T* = ±1 ◦C. In order to determine fracture toughness characteristics, force *P*, deflection of the specimens (sensor 3) and crack opening displacement (*COD*) (sensor 4) were recorded while the load was applied to the sample. Acoustic emission signals were recorded using a 24 channel μSAMOS acoustic emission processor with the AEwin and NOESIS 12.0 software developed by the Physical Acoustics Corporation (PAC) from the Princeton, NJ, USA. Two low-frequency sensors with a flat response curve in the 30–80 kHz range (VS30-SIC-40dB, manufactured by Vallen GmbH) (sensor 5) and two broadband sensors with a frequency range of 100–1200 kHz (WD 100–1200 kHz, manufactured by PAC) (sensor 6) were used to record AE signals in a broad frequency range (Figure 1b).

**Figure 1.** The SENB specimen in three-point bending with installed AE sensors: (**a**) photograph of the specimen in the thermal chamber; (**b**) scheme of loading and installation of mechanical and AE sensors.

**Table 1.** Mean values of the mechanical properties of AA2519 aluminium alloy and Ti6Al4V titanium alloy at di fferent temperature.

AE parameters were clustered with the k-means method in a Euclidean space. This method involves the iterative search for the set of reference elements representing the individual clusters. Successive approximations of the reference elements are sought in each iteration through calculations using the indicated methods. Depending on the adopted assumptions, the reference element may be one of the elements of the *X* population or an element of a specific set *U* ⊇ *X*. In metric spaces, the reference element may be calculated as an arithmetic mean, and it accordingly represents the centre of gravity of the cluster.

In general, the input for the k-clustering algorithm is the set of objects *X* and the expected number of clusters *k* and the output is the division into subsets *{C1, C2,. . . , Ck}*. Usually, k-clustering algorithms belong to the category of optimisation algorithms. Optimisation algorithms assume that there is a loss function *k*: *{x*|*X* ⊆ *S}* → *R*+ defined for every *S* subset. The purpose is to find the group with a minimal sum of the losses described by the following Formula (1):

$$E\_{\emptyset} = \sum\_{i=1}^{k} k(\mathbb{C}\_{i}) \tag{1}$$

In order to use the iterative algorithm, it is necessary to determine the dissimilarity measure used in the clustering process. In this case, it will be a point that is the resultant point for the particular cluster (representative element) calculated, for instance, as the geometric or arithmetic mean, described with the following Formula (2):

$$k(\mathbf{C}\_i) = \sum\_{r=1}^{|\mathbf{C}\_i|} d\left(\overline{\mathbf{x}}^i, \ \mathbf{x}\_r^i\right) \tag{2}$$

where: *xi*- arithmetic (or geometric) mean of the cluster.

The most important advantage of this method is the speed of data processing and analysis.

The research also used other grouping methods (Forgy, Normalized Normal Constraint (NNC), Fuzzy C-Means (FCM), Gaussian Mixture Decomposition (GMD), Gustafson-Kessel (GK), Hidden Markov Model (HMM), Autoregressive Hidden Markov Model (ARHMM)) to isolate destructive subprocesses based on a full statistical-mathematical approach to divide into groups [37–41].

Analyses using waveform time domain, waveform time domain (autocorrelation), fast Fourier transform (FFT Real) and waveform continuous wavelet (CWT) based on the Morlet wavelet were also conducted in order to verify the divisions made using the k-means method.

The waveform time domain chart shows a change of amplitude expressed in units of voltage V within a specific period. It is also useful in determining the rise time or duration of the acoustic signals for the indicated AE signal classes. The waveform time domain (autocorrelation) chart, in turn, is significant to technical diagnostics and to signal processing and transmission theory. The autocorrelation function is used to determine the rate of signal change and to detect periodic

signals in "noisy" measurement signals—which is very important to the processing of diagnostic signals. The autocorrelation function ϕ*x*(τ) of signal *x(t)* with limited energy is described by the following Formula (3):

$$\varphi \chi(\tau) = \int\_{-\infty}^{\infty} \mathfrak{x}(t) \mathfrak{x}^0(t-\tau) dt \tag{3}$$

The value of autocorrelation function ϕ*x*(τ) at point τ = *0* is real, and it is equal to the energy of the *x(t)* signal described with the following Formula (4):

$$\varphi\_{\mathcal{X}}(\pi) = \int\_{-\infty}^{\infty} x |\mathbf{x}(t)|^2 dt = E\_{\mathcal{X}} \tag{4}$$

This property can be derived directly from Formula (3) after substituting τ = *0*.

The charts of the fast Fourier transform (FFT Real) can be used for an analysis in the frequency domain to identify the key frequencies in the entire data set instead of examining every change in the time domain. The chart in the frequency domain shows the phase shift or strength of the signal at each frequency on which it exists. It shows how much signal is contained in the particular frequency band within a specific frequency range.

Important frequency and energy-related information of AE signals can be extracted with the waveform continuous wavelet chart based on the Morlet wavelet. Waveform continuous wavelet (CWT) in the time domain is described by the following Formula (5):

$$\mathcal{CNT}(t,a) = \frac{1}{\sqrt{|a|}} \int\_{-\infty}^{\infty} x(\tau) \chi\left(\frac{\tau - t}{a}\right) d\tau \tag{5}$$

where *t*—time instant of the tested signal*;x(*τ*)*—analysed signal; γ *(t)*—base filter function, so-called mother function; *a*—scale of the mother function.

The features extracted from this wavelet transform from can be significant to the detection of structural damage. By using a suitable family of the base signal filter function (so-called wavelet mother), it is possible to identify a temporary variation of the analysed signal. Another advantage of using *CWT* is the detection of the variation of the analysed value and/or—depending on the domain of the analysed response—identification of the location or time of damage. The Morlet wavelet was selected as the mother function for the analysis of the response signal obtained through the simulation of damage. The Morlet wavelet is currently used in various applications including successful use in damage diagnostics.

### **3. Results of Experimental Tests**

The critical values of fracture toughness were calculated as *J*-integral at the point of reaching the maximum force using the Rice formula: *J*C = 2*A*C/*B*(*W* – *a*0), where *A*C is the energy absorbed by plastic deformation and crack growth in the sample. For room temperature, *T*1 = 20 ◦C, the integral in accordance with the Rice formula reached *J*C = 46 kN/m, and as the test temperature lowered, *T*2 = −50 ◦C, the critical value of *J*-integral decreased slightly, reaching *J*C = 40 kN/m, which was consistent with expectations. For metals, particularly ferritic steels, a decrease of the test temperature will reduce critical fracture toughness characteristics [42–46].

However, careful observation and comparison of the *P*–*COD* load curves with the recorded AE signals indicates certain peculiarities of the fracture process in Al–Ti laminate specimens tested at two temperatures: *T*1 = 20 ◦C and *T*2 = −50 ◦C (Figure 2). For the lowered temperature, the *P*–*COD* curve has slightly lower values than for room temperature. It should be emphasised that in samples made from homogeneous materials, Al (AA2519) and Ti (Ti6Al4V) alloys [15], and in specimens from

steel [15,47], an opposite trend could be observed—reducing the temperature increased the strength characteristics and Young's modulus, and it made the rise of the chart in the loading section accordingly steeper. Also, in the rising sections of the load curves, we can observe a deviation from linearity occurring when the force is approximately 4 kN for the specimens tested at temperature *T*2 = −50 ◦C and approximately 6 kN for tests at temperature *T*1 = 20 ◦C.

The distributions of AE signals in both samples (Figure 2) clearly show that signals with a high strength (above 5.0 × 107 pV·s) appear when the force is decreasing, directly after reaching the maximum force levels which corresponds to the start of the main subcritical crack. Also, the AE signals with high strength for the sample tested at *T*2 = −50 ◦C were located primarily in short time sections just after the maximum force level, which indicates a rapid growth of the subcritical crack. For the sample tested at *T*1 = 20 ◦C, AE signals with high strength also appeared while the load was being applied which indicates a continuous development of the subcritical crack.

**Figure 2.** Load curves of the specimens with AE signals. (**a**) *T*1 = 20 ◦C; (**b**) *T*2 = −50 ◦C.

AE signals for which signal strength (SS) was within the range of 1.0 × 107–5.0 × 107 pV·s were very significant to the assessment of the generated destructive processes in the tested samples. These signals appeared in various phases of the loading of the samples tested at *T*2 = −50 ◦C and *T*1 = 20 ◦C. At room temperature, most of these signals were recorded along the falling part of the *P*–*COD* load curve, representing the increase of subcritical crack. However, during the test at lowered temperature (*T*2 = −50 ◦C), the great majority of signals from that range were recorded in the specimen loading phase before the maximum force was reached, i.e., when the subcritical crack did not start yet. The observed difference in the appearance of signals from that range may indicate that the Al–Ti laminate fracture process develops differently at different temperatures. This assumption is also supported by the peculiarities observed in the *P*–*COD* curves as indicated above.

Fracture surfaces of the samples were observed with a scanning electron microscope (SEM), and the recorded AE signals were analysed using a non-hierarchical method for AE signal clustering based on *k*-means [30,31] and analysis using waveform time domain, waveform time domain (autocorrelation), fast Fourier transform (FFT Real) and waveform continuous wavelet based on the Morlet wavelet [32] in order to explain the peculiarities of the *P*–*COD* curves at different temperatures and identify the mechanisms generating the AE signals.

### **4. Tests of Fracture Surfaces and Microstructure Using SEM**

Observations of the fracture surfaces of the tested specimens using an optical microscope and SEM clearly indicated the formation and development of two mutually perpendicular cracks—the main crack, which developed from the pre-crack in the same plane, and the delamination crack, which developed in a perpendicular plane between the component layers of Ti and Al alloys (Figure 3). The presence of delamination cracks was observed in all samples tested at both temperatures. In most cases, the delamination crack was formed on the Ti6Al4V alloy side, but there were also situations where the crack was formed at the AA2519 alloy layer, or even in parallel on both sides next to the base layers (Figure 3b).

**Figure 3.** Delamination crack: (**a**) total view of fracture surface of the SENB specimen; (**b**) development of the delamination crack.

The nature of the delamination crack clearly indicates the presence of two mechanisms in the development of the fracture—brittle phase fracture and shear in the AA1050 aluminium alloy (Figure 4). Most likely, a brittle fracture may be initiated by the brittle fracture of the particles of Al and Ti intermetallic compounds [45] or oxides formed in the transition zone (TZ) between the Al alloy (AA1050) and Ti alloy (Ti6Al4V) during explosion welding (Figure 5). The presence of elements of metals and oxygen was confirmed through EDS analysis in the transition zone (Figure 6, Table 2). In the photographs (Figure 4), the areas of the fractured brittle phases are clearly visible as the light areas from which shear develops in the Al alloy (AA1050). In samples tested at temperature *T*1 = 20 ◦C, the growth of the delamination crack occurred primarily by shear (Figure 4a), and at the reduced temperature, *T*2 = −50 ◦C, there were large areas with brittle fracture (Figure 4b).

(**a**)  **4.**

*Cont.*

**Figure**

**Figure 4.** Nature of the delamination crack. (**a**) tests at *T*1 = 20 ◦C; (**b**) tests at *T*2 = −50 ◦C.

Observations of metallographic photographs of the connecting zone of the base materials (Figure 5) indicated that this narrow strip with a width of up to 40 μm (transition zone—TZ) between the material of the inner layer, AA1050 alloy, and the Ti6Al4V alloy (Figure 5b), was the weakest link in the Al–Ti laminate. This is precisely the strip formed by the mutual penetration of materials during explosion bonding. The appearance of brittle fracture in that zone is caused by different types of particles and material discontinuities present in the TZ.

**(a)**

**Figure 5.** *Cont.*

**Figure 5.** (**a**) inner layer and connecting zone of the base materials; (**b**) transition zone (TZ) between the AA1050 alloy and the Ti6Al4V alloy.

The complementary tests conducted using the EDS analysis showed that the TZ contained elements of metals from the base layers (Figure 6a), which formed Al and Ti intermetallic compounds [47], and oxygen (Figure 6b), which indicated the existence of metal oxides (Table 2).

**Figure 6.** The point of EDS analysis in TZ in aria 1 (**a**) and aria 2 (**b**).

**Table 2.** The results of EDS analysis.

The development of the main crack, which grew from the pre-crack in the same plane, occurred in both alloys: Al (AA2519) and Ti (Ti6Al4V), in accordance with the ductile mechanism through the formation and connection of voids. However, there are certain noticeable differences. In the Al alloy (AA2519), voids with a size of 10–20 μm are formed around fairly large particles with a size of 2–5 μm (Figure 7a). The morphology of the fracture surface of the Ti (Ti6Al4V) alloy, in turn, consists of smaller fragments of voids with a size of 2–7 μm (Figure 7b). The nature of the propagation of the main crack is similar in the samples tested at both temperatures.

 **Figure 7.** Fracture mechanism in the base materials. (**a**) Al alloy (AA2519); (**b**) Ti alloy (Ti6Al4V).

### **5. Analysis of AE Signals**

The AE signals recorded during loading of the specimens at *T*1 = 20 ◦C and at *T*2 = −50 ◦C (Figure 2) were preliminarily clustered using the non-hierarchical method for AE signal clustering—k-means. As a result of the clustering, the signals were divided into five classes shown on point charts of AE signal strength (SS) pV·s, over time (s) (Figure 8).

**Figure 8.** Point charts of signal strength (SS) over time with breakdown into classes describing the characteristic fracture mechanisms. (**a**) for *T*1 = 20 ◦C; (**b**) for *T*2 = −50 ◦C.

The charts also show the force acting on the sample, P (kN), which was recorded with a set of acoustic instruments only when the AE signals were present. Since the sample was loaded and partially unloaded, the AE signals intensify when the load is rising and diminish as the load is decreasing.

It is to notice that in the sample tested at *T*1 = 20 ◦C (Figure 8a), the great majority of AE signals from classes 1–5 occurred just after the maximum force was reached and later, i.e. after the main crack has propagated. When the load was rising, only AE signals with low signal strength (SS) were recorded—for class 1, the SS reached 8.0 × 106 pV·s, and, for class 2, the SS values were within the range of 4.8 × 106 to 2.4 × 107 pV·s.

During tests at temperature *T*2 = −50 ◦C (Figure 8b), in the rising part of the load curve, up to the point at which maximum force was reached, numerous signals of classes 1 to 4 were recorded, which indicates an intense growth of the crack during that period. Just after the maximum force was reached, AE signals of classes 1 to 5 were recorded, with no signals of classes 2–5 being recorded after the force dropped below 0.68 Pmax (less than 5.3 kN).

The preliminary clustering of AE signals using the k-means method was followed by an in-depth analysis of AE signals using the following charts: waveform time domain (WTD) (Figure 9), fast Fourier transform (FFT Real) and waveform continuous wavelet using the Morlet wavelet (Figures 10–14) and waveform time domain (autocorrelation)—complex Fourier series (Figure 15).

The preliminary clustering of AE signals using the *k*-means method was followed by an in-depth analysis of AE signals using the following charts: (WTD) (Figure 9), waveform frequency domain (Real)—FFT Real and waveform continuous wavelet using the Morlet wavelet (Figures 10–14) and waveform time domain (autocorrelation)—complex Fourier series (Figure 15). The charts shown in Figures 9–15 can be used to verify and adjust the preliminary clustering of AE signals. In Table 3 are presented ranges and maximum levels of AE signal characteristics obtained by this analysis.

**Table 3.** Ranges and maximum levels of AE signal characteristics.

Class-5 signals (red points) are generated in connection with the ductile development of the main crack by the growth of voids, which is driven by the dislocation motion. They are characterised by a high amplitude of up to 8.81 V and low frequency level in the two dominant bands (Figure 14a,b): 60 and 70 kHz. The 60 kHz frequency is characteristic to aluminium alloys, and the frequency of 75 kHz characterises titanium alloys. These processes related to the growth of voids have long durations of up to 4000 μs (Figure 9e), high energy values of up to 12,000 eu (Figure 15e) and signal strength from 8.8 × 107 to 1.2 × 108 pV·s (Table 3).

Class-4 signals (Bottle Green points) also include two characteristic frequency ranges: approx. 60 kHz and 250–300 kHz. The structure of the class-4 signal indicates that it is generated in the shear process, plastic deformations appear first with a frequency of approximately 60 kHz, followed by shear fracture characterised by a frequency of 250–270 kHz (Figure 13a,b). These signals are accompanied by a high amplitude, reaching 8.8 V and duration of up to 1000 μs (Figure 9d). The signal strength for class-4 signal ranges from 3.0 × 107 to 5.8 × 107 pV·s, and the signal energy calculated from the WTD reaches up to 18,000 eu (Figure 15d).

Class-2 (dark blue) and class-3 (pink) AE signals are characterised by a similar frequency in the range of 250–270 kHz and duration of approximately 520 μs, but they di ffer in amplitude, signal strength and energy (Table 3).

**Figure 9.** *Cont.*

**Figure 9.** Waveform time domain charts for the individual classes of AE signals. (**a**) class 1; (**b**) class 2; (**c**) class 3; (**d**) class 4; (**e**) class 5.

**Figure 10.** *Cont.*

**Figure 10.** Waveform frequency domain (Real) (**a**) and waveform continuous wavelet (Morlet) (**b**) charts for the individual classes of AE signals (class 1).

**Figure 11.** Waveform frequency domain (Real) (**a**) and waveform continuous wavelet (Morlet) (**b**) charts for the individual classes of AE signals (class 2).

A higher amplitude, reaching up to 8.8 V could be observed with class-3 signals. These signals reached signal strength in the range from 1.1 × 107 to 4.6 × 107 pV·s, and their maximum energy calculated from the WTD was 11,500 eu (Figure 9c, Figure 12a,b and Figure 15c).

Class-2 signals were characterised by lower signal strength, in the range from 4.8 × 106 to 2.4 × 107 pV·s and 3.5 times lower energy, not exceeding 3000 eu (Figure 9b, Figure 11a,b and Figure 15b), determined through the WTD analysis. Both types of signals were generated by the brittle fracture mechanism. Most likely, they characterise brittle phase fracture in the transition zone: class-3 signals—fracture of Al–Ti intermetallic compounds, whereas class-2 signals indicate oxide fracture.

Class-1 (Light green) AE signals with a frequency of approximately 100 kHz had a very low amplitude, not exceeding 0.4 V, and they were characterised by signal strength in the range from 1.8 × 104 to 8.0 × 106 pV·s and energy of up to 10 eu determined through the WTD analysis (Table 3, Figure 9a, Figure 10a,b and Figure 15a).

**Figure 12.** Waveform drequency domain (Real) (**a**) and waveform continuous wavelet (Morlet) (**b**) charts for the individual classes of AE signals (class 3).

These signals were generated by "noise", and they were usually caused by the friction of the rollers against the sample or by the operation of the strength testing machine. Class-1 signals will not be considered in further analysis of the fracture process.

**Figure 13.** Waveform frequency domain (Real) (**a**) and waveform continuous wavelet (Morlet) (**b**) charts for the individual classes of AE signals (class 4).

**Figure 14.** *Cont.*

**Figure 14.** Waveform frequency domain (Real) (**a**) and waveform continuous wavelet (Morlet) (**b**) charts for the individual classes of AE signals (class 5).

**Figure 15.** *Cont.*

**Figure 15.** Waveform time domain (autocorrelation) chart for the individual classes of AE signals. (**a**) class 1; (**b**) class 2; (**c**) class 3; (**d**) class 4; (**e**) class 5.


*2.3. Characterizations*

The surface morphologies of the 3D nanostructures were characterized by field emission scanning electron microscopy (FE-SEM; JSM-6700F, Joel, Tyoko, Japan) and transmission electron microscopy (TEM; JEM-2100F, Joel, Tyoko, Japan), respectively. Diffuse reflectance spectra were measured using a UV-Vis-NIR spectrophotometer (Cary 5000, Agilent Technology, Santa Clara, CA, USA). Visible light is mainly reflected by glass microfibers; therefore, the extinction spectra were converted directly from the diffuse reflectance spectra. The SERS spectra were measured using a handheld Raman spectrometer (CBEx, Snowy Range Instruments, Laramie, WY, USA) equipped with 785 nm and 638 nm lasers. The incident laser power and exposure time were 10 mW and 0.01 s for the SERS measurements, respectively.

## *2.4. Numerical Simulations*

Numerical simulations were carried out using finite difference time domain analysis (FDTD) techniques. In these simulations, transverse magnetic polarized incident waves with different wavelengths (450 nm, 532 nm, 633 nm, and 785 nm, respectively) were applied to the crossed Ag NW nanostructure. The simulation was simplified by including three crossed Ag NWs (the upper and lower NWs were perpendicular to the central NW). The Ag NWs were modeled as five-sided equilateral pentagons, and the radius of the pentagon was fixed at 25 nm. The mesh size was set to 0.2 nm around the junction of the crossed Ag NWs. Perfectly matched layer (PML) formulation was applied in all directions. The complex permittivity of Ag was adopted from Reference [19].

#### **3. Results and Discussion**

#### *3.1. Compact Integration of the TiO2 NPs into the 3D Cross-Points of Vertically Stacked Ag NWs*

3D hybrid TiO2/Ag NW structures were fabricated using a simple two-step vacuum filtration method. First, 3D-stacked Ag NWs were prepared by vacuum filtering 4 mL of a dispersion containing 0.5 wt% Ag NWs over a glass microfiber filter [20,21]. Then, 4 mL of an aqueous solution containing TiO2 NPs were poured over the 3D stacked Ag NWs without breaking the vacuum. Figure 1a shows a scanning electron microscopy (SEM) image of the 3D stacked Ag NWs. Within the 3D multilayered Ag NWs, small nanogaps formed at the 3D cross-points at which the NWs were vertically stacked. Transmission electron microscopy (TEM) images clearly revealed that each Ag NW assumed a pentagonal shape, and small nanogap regions formed at the 3D cross-points, at which the lower and upper layers were closely spaced (dotted rectangle in Figure 1b). As small pores formed in the 3D crossed region, 22 nm (average diameter) TiO2 NPs were selectively filtered during the vacuum filtration process. Figure 1c,d show the 3D hybrid nanostructures consisting of TiO2 NPs and 3D Ag NWs upon application of a 0.002 wt% TiO2 NP solution to the 3D stacked Ag NWs. The small TiO2 NPs were mainly filtered from the solution and remained on top of the 3D cross-points regions, resulting in the compact integration of TiO2 NPs into the 3D hot spot region. These interfaces enhanced the interactions between Ag NWs and the TiO2 NPs. The 3D stacked AgNWs could be also considered as photocatalytic membranes [22,23]. In addition, the compact hybrid nanostructures embedded on glass microfiber filter would be easily separated from treated solution, which can be applied in water treatment [24,25]. The TiO2 NPs covered the entire 3D surface of the Ag NWs when a 0.01 wt% TiO2 NP aqueous solution was used (Figure S1).

**Figure 1.** (**a**) SEM and (**b**) TEM images of 3D stacked Ag NWs. (**c**,**d**) SEM images of 3D hybrid nanostructures consisting of TiO2 NPs and 3D Ag NWs. The image clearly shows the compact integration of TiO2 NPs onto the crossed region of the 3D Ag NWs.

#### *3.2. Optical Properties and Local Field Enhancements of the 3D Hybrid Nanostructures*

Figure 2 presents the extinction spectra of 3D plasmonic Ag NWs and 3D TiO2/Ag hybrid nanostructures prepared with different TiO2 concentrations (see Figure S2 for the extinction spectra of the bare microfiber filters and TiO2 NPs). The microfiber substrate deposited using 0.01 wt% TiO2 NPs exhibited a very low extinction in the visible region but a very high UV extinction below 400 nm. These results indicated the presence of UV light–TiO2 semiconductor interactions near the band gap of crystalline TiO2 [26]. The green tangent line indicates 388 nm, the band gap energy (3.2 eV) of the anatase TiO2 (Figure S2). The 3D Ag NWs exhibited strong interactions from the UV to the visible region (black line in Figure 2). The highest extinction near 326 nm was attributed to an interband transition (3.8 eV) of Ag. The 3D stacked Ag NWs also had a high extinction across the entire visible wavelength range. The 3D hybrid nanostructures possessed higher extinction than the 3D stacked Ag NWs in visible region. The high extinction of the 3D hybrid nanostructures agreed well with the high intensity of visible light under standard AM 1.5G solar illumination (navy line in Figure 2). Therefore, the 3D hybrid nanostructures were expected to increase the photocatalytic activities through enhanced light–matter interactions.

**Figure 2.** Extinction spectra of different nanomaterials and spectral irradiance of AM 1.5G. The composite nanostructures displayed high extinction in the visible range, in agreement with the solar spectrum in the visible range. The black lines indicate the interband transitions of Ag.

Another prominent property of the 3D plasmonic or hybrid nanostructures is the local field enhancement due to a coupled LSPR effect at the 3D cross-points [27–31]. Figure 3 shows the near-field enhancement distribution contours of orthogonal Ag NWs (similar to the Ag NW stack indicated by the white dotted area in Figure 1b) for different incident wavelengths obtained from finite-difference time-domain (FDTD) simulations (Figure S3). The strong field was localized and confined at the nanogap region of the cross-points of vertically stacked Ag NWs, regardless of the wavelength of incident light. The field intensity enhancement could be further increased by increasing the curvature of the metal nanostructures due to a lightning rod effect, indicating that the pentagonal shape provided stronger enhancement than the circular shape [32]. The maximum field enhancement was obtained at the bottom corners of the central pentagonal Ag NW. The maximum field enhancement increased by an order of magnitude at shorter wavelengths (Table 1). The average field enhancement along the middle Ag NW surface could be extracted from the simulation results (Table 1). Note that we extracted the field enhancement value 0.5 nm away from the Ag NW surface to avoid any staircase effects in the simulation (green lines in Figure S4). The average field enhancement corresponded to the maximum field enhancement (Table 1). The numerical simulations suggested that a highly enhanced local field could be generated at the cross-points of the 3D stacked Ag NWs. Therefore, it is important to deposit TiO2 NPs selectively onto these hot spot regions to ensure efficient charge separation and enhanced photocatalysis.

**Figure 3.** Finite difference time domain (FDTD) simulations of the electric field distribution (|E|2/|E0|2) at the junctions of crossed Ag NWs, using the wavelengths (**a**) 450 nm, (**b**) 532 nm, (**c**) 633 nm, and (**d**) 785 nm. The k and E vectors in the figure show the incident direction and the polarization direction of the illumination, respectively. The white lines are used to indicate the boundaries of Ag NWs. All scale bars indicate 20 nm.

**Table 1.** Maximum and average field enhancement of crossed Ag NWs at different wavelengths.

## *3.3. Photocatalytic Performance*

The photocatalytic activities of various nanomaterials were examined using the photodegradation of an organic dye (methylene blue (MB)). The photocatalytic degradation was carried out under AM 1.5G (or 1 sun) simulated illumination with an intensity of 100 mW/cm2, which are standard light illumination conditions for measuring solar cell efficiency [33]. This standard light illumination provided a quantitative photocatalyst performance comparison, unlike other light sources, such as high-pressure mercury lamps with a high power [34]. All photocatalysts were independently examined in the presence of 10 mL of a 0.05 mM MB aqueous solution under 10 min illumination. Figure 4a shows the absorbance changes of an MB solution after 10 min light illumination of each photocatalytic substrate. The main absorbance peak of MB, at 665 nm, was found to decrease in intensity after light illumination of each photocatalyst. Negligible MB degradation was observed during 10 min illumination in the absence of a substrate, indicating that MB itself did not decompose under illumination. Interestingly, the 3D stacked plasmonic Ag NWs exhibited higher photocatalytic activities compared to the TiO2 NPs, primarily due to LSPR-induced hot electron generation and the direct decomposition of MB [35,36]. The 3D hybrid nanostructures composed of 0.002 wt% TiO2 NPs and 3D stacked Ag NWs showed the highest solution MB absorbance changes in the UV-visible and visible ranges (Figure 4b). At higher concentrations (i.e., 0.01wt% TiO2 NP solution) of TiO2, the photocatalytic activities decreased due to inefficient charge separation at the Ag NWs and TiO2 interface and blockage of MB molecular mass transport into the hot spot regions by the large surface coverage of TiO2 NPs (Figure S1). The photocatalytic activities of the different nanostructures were examined by calculating the decrease in MB absorbance after illumination (Table 2). The TiO2 photocatalyst was active only under UV illumination due to its large band gap. No MB degradation was observed under visible illumination in the presence of the TiO2 photocatalyst (Figure S5). The stacked 3D Ag NWs showed better activities in both the UV-visible and visible ranges compared to the TiO2 NPs. The optically excited plasmonic nanostructures activated the chemical transformations directly on their surfaces [37–40]. Among the TiO2/Ag NW composite nanomaterials, the composite prepared with 0.002 wt% TiO2 NPs exhibited the best MB photocatalytic activities under both spectral ranges. In the presence of the 3D hybrid nanostructures, the MB concentration degraded to one-half of its original value after only 10 min illumination.

**Figure 4.** Photocatalytic performances of 0.05 mM methylene blue (MB) aqueous solutions after 10 min light illumination in the presences of different photocatalysts under (**a**) AM 1.5G simulated illumination and (**b**) visible and near-infrared illumination (λ > 400 nm).

The 3D composite nanostructures composed of 0.002 wt% TiO2 and 3D Ag NWs were examined in time-dependent MB degradation studies under different light illumination conditions. The changes in the UV-visible spectra during photodegradation are shown in Figure 5. The main absorption peak of MB at 665 nm decreased markedly with the irradiation time (up to 60 min) and almost vanished after 90 min (Figure 5a). After 90 min of irradiation, the blue color of the initial MB solution became transparent due to the photodecomposition of MB molecules. We observed almost the same photocatalytic performance in the presence of the 3D composite under visible light illumination (λ > 400 nm), as shown in Figure 5b. The 3D composite performed effectively under both full spectral and visible spectral (λ > 400 nm) illumination.

**Figure 5.** Time-dependent UV-vis spectra of an MB aqueous solution after illumination in the presence of 0.002 wt% TiO2/Ag NW composites under (**a**) UV-visible light and (**b**) visible light.

**Table 2.** The photocatalytic efficiencies of different photocatalysts under AM 1.5G and visible and NIR (the UV was cut off by a filter) light after 10 min illumination\*.

\* 10 mL 0.05 mM MB was used to test the photodegradation of organic dyes during illumination.

The photocatalytic efficiencies of the 3D composite nanomaterials are illustrated in Figure 6. *C*0 is the initial concentration of MB, and *C* is its concentration after each irradiation time. About 90% of the initial MB concentration was photodegraded under UV/visible and visible illumination after 60 min. The rate of MB degradation decreased after 60 min. This decrease was attributed to the fact that the substrate was placed at the bottom of a petri dish, and the MB molecules in the aqueous solution were randomly dispersed throughout the 10 mL volume during illumination. The dilution of intact MB molecules decreased the rate of collision between the organic molecules and the active site. The diffusion-limited behavior of the dye molecules required longer illumination times to achieve total decomposition of the organic dyes. Due to the diffusion-limited behavior, we divided the photocatalytic kinetics into two stages: before 60 min and after 60 min. The photodegradation process was assumed to follow Langmuir–Hinshelwood kinetics, *ln* (*C*/*C*0) = −*kt*, where *C* is the MB concentration as a function of the photodegradation time (*t*), *C*0 is the initial concentration, and *k* is the reaction rate constant (insets of Figure 6) [41,42]. The rate constant obtained under UV-visible illumination was measured to be 3.893 × 10−2 min−1 during the initial stage and decreased to 1.438 × 10−2 min−1 after 60 min (Inset of Figure 6a). Under visible light illumination, the values of *k* were found to be 3.876 × 10−2 min−1 and 1.084 × 10−2 min−1, respectively (inset of Figure 6b).

**Figure 6.** Photodegradation of an MB aqueous solution in the presence of 0.002 wt% TiO2/Ag NW composite nanomaterials under (**a**) UV-visible light and (**b**) visible light. The inset figures show the linear fits to *ln*(*C*/*C*0) vs. time.

#### *3.4. Mechanism Underlying the Plasmon-Enhanced Photocatalysis*

Semiconductor TiO2 NPs are typically integrated with 3D plasmonic Ag NWs with the goal of using the LSPR properties of 3D plasmonic Ag NWs to enhance visible light absorption and channel hot electrons to the TiO2 NPs for efficient photocatalysis, a plasmonic sensitization process [17]. In 3D hybrid nanostructures, a Schottky barrier forms at the junction interface between Ag and TiO2 (Figure 7). This barrier blocks electron transfer from the Ag NWs to the TiO2 NPs. Upon excitation of the LSPR, highly energetic electrons (hot electrons) have sufficient energy to overcome the Schottky barrier, and they can inject into the conduction band of the TiO2. The plasmonic sensitization process shuttles extra electrons to the wide-band gap (3.2 eV) TiO2, which is originally inactive under visible light. The TiO2 can then perform catalytic reduction reactions [5,17]. The holes left behind in the Ag NWs can be used to oxidize organic molecules. As most of the electron/hole pairs are produced in the field-enhanced region of the 3D crossing points of vertically stacked Ag NWs (Figure 3), efficient charge separation occurs at the interface between the 3D cross-points and nearby TiO2 NPs. Therefore, the compact integration of crystalline TiO2 NPs into the 3D cross-points can enhance the photocatalytic performance over the full spectral range.

**Figure 7.** Schematic band energy diagram of charge transfer at the Ag NW/TiO2 NP interface (not drawn to scale). CB and VB indicate the conduction band and valence band of anatase TiO2. EF presents the Fermi level of Ag. ΦB is the Schottky barrier height.

#### *3.5. Spectroscopic Investigation of Plasmon-Enhanced Photocatalysis*

We collected the Raman spectra of organic dye molecules to investigate their plasmon-enhanced photocatalytic degradation by 3D stacked Ag NWs and 3D hybrid nanostructures. First, 3 μL of a 5 μM MB aqueous solution was applied to each photocatalytic substrate. After drying, SERS spectra were

obtained using a portable Raman spectrometer [21,43]. The SERS measurements were collected using a low-power 10 mW illumination beam with a spot size of 20 μm incident on each substrate for a very short laser exposure time of 0.01 s to avoid photocatalytic degradation of the MB molecules. The laser was then directed onto the same spot on the substrate for 10 s to induce photocatalytic degradation of the MB molecules. SERS measurements were performed to detect any SERS intensity changes in the probe molecules. Each cycle consisted of a photodegradation process (10 s illumination) and a SERS measurement (0.01 s illumination of 10 mW laser). Figure 8 presents the SERS intensity changes in MB molecules on the 3D stacked Ag NWs (Figure 8a) and composite substrate (Figure 8b) as the cycle time was increased with laser illumination at 638 nm and 20 mW. Prior to the photodegradation process, a high-intensity characteristic MB Raman spectrum was obtained from the 3D stacked Ag NWs (black line in Figure 8a) using 10 mW 638 nm laser illumination over 0.01 s. Because the 3D stacked Ag NWs had a high density of hot spots (crossed regions between Ag NWs) for use in SERS [18,21,27,44], we obtained highly enhanced MB SERS spectra from the 3D Ag NWs. The SERS intensity of MB decreased significantly after the first photodegradation process (10 s illumination at 20 mW, red line in Figure 8a). The SERS intensity remained unchanged during additional photodegradation cycles. We performed these cyclic photodegradation and SERS measurements using a 3D composite substrate (Figure 8b). Interestingly, the initial SERS intensity of the MB obtained from the 3D composite substrate increased beyond 40%, compared to the spectrum obtained from the 3D stacked Ag NWs. This increase was attributed to the compact integration of 3D TiO2 NPs on the crossed regions of the Ag NWs. Because 3D volumetric hot spots formed, more MB molecules could be deposited onto the TiO2 NPs and Ag NWs within the 3D hot spot volume [43]. The MB SERS intensity decreased significantly after only one photodegradation process (10 s illumination at 20 mW, red line in Figure 8b). Plasmonic metal nanostructures strongly absorb the light, leading to significant local heating associated with the resonant plasmonic excitations [45,46]. This thermoplasmonic effect, therefore, partially enabled the plasmon-induced reactions, particularly in the cross-point regions of Ag NWs [47].

**Figure 8.** SERS intensity changes in the MB deposited onto (**a**) 3D stacked Ag NWs structures and (**b**) 3D hybrid (0.002 wt% TiO2/Ag NWs) nanostructures during 638 nm laser exposure (10 s). The SERS measurements were performed by illuminating with a 638 nm laser for only 0.01 s to avoid photocatalytic effects. (**c**) The overall SERS intensity changes at 1620 cm−1 in MB molecules under different illumination conditions.

Figure 8c presents the overall SERS intensity changes at 1620 cm−1 of MB molecules under different illumination conditions. Figure 8c reveals several important characteristics of the plasmon-enhanced photocatalysis reaction. First, the 3D stacked Ag NWs displayed higher photocatalytic activities at shorter illumination wavelengths, in agreement with simulation results of local field enhancements and the optical extinction spectra of 3D plasmonic Ag NWs. Second, for a given photocatalyst and incident wavelength of light, increasing the incident light intensity increased the photocatalytic activity [48]. These results indicated that a quantitative comparison of the photocatalytic activities of the different catalysts required fixed light irradiation conditions, including the light source and intensity. Third, the 3D composite structures exhibited superior photocatalytic activities over the 3D Ag NWs, in good agreement with the photocatalytic performances. Finally, the photodegradation process mainly occurred at specific sites. We clearly observed that the SERS intensities of the probe molecules dramatically decreased only during the first cycle, then remained unchanged during the additional nine cycles. This result was attributed to the fact that prior to the photodegradation process, MB molecules deposited onto the crossed regions mainly accounted for the high SERS intensity. MB molecules on the hot spots decomposed during the first photodegradation process, and the SERS intensity decreased significantly due to the absence of target molecules in the hot spot. MB molecules deposited onto the non-crossed regions did not decompose, even after ten cycles, and MB molecules in non-hot spot regions (such as Ag NWs at the non-crossed region) contributed to a low SERS intensity. That is, hot spots were cross-points in the 3D stacked Ag NWs and hybrid TiO2/Ag NWs structures for the SERS and photodegradation process. The spectroscopic studies of plasmon-enhanced photocatalysis revealed that excess irradiation onto hot spot areas could trigger the photo-induced elimination of target molecules, reducing the sensitivity (or increasing the limit of detection) in highly sensitive plasmonic nanostructures during SERS measurements.


**6. Prophylaxis and treatment of BONJ**

Recently inIn recent literature is found date for, a new type of complication associated with bisphosphonate intake –has been found, which is called avascular necrosis of the jaws. It is defined as necrosis associated with or without dental procedures, which could persist for more than 6-8 weeks with irresponsive to conservative treatment, found in patients without history of previous radiotherapy in the area affected but treated with nitrogen group bisphosphonates with nitrogen group, i.v. through IV for at least a year or per os for longer period, and associated with general condition withof bone resorption. Similar cases arewere published for the first time in 2003 by Marx with patients treated with pamidronate and zoledronate. Later, Carter and Gross, Ruggierro, Migliorati, and Wang reportalso reported similar cases. In 2005, Novartis (Aredia and Zometa48 producer) officially declaredeclared 475 cases of Bisphosph‐ onate bisphosphonate-related osteonecrosis of the jaws. In nowadaysNowadays, the number of affected patients worldwide is unknown. Scientific literatures says the BONJ is from 1,.3% to 10%. More There are more than 5,.1 million patients are treated with bisphosphonates. More and more than 2 million acceptaccepted BPs as antimalignatanti-malignant therapy. The number of patients treated for osteoporosis with BPs is increasing rapidly. More than 10 million patients, mainly women in the USA has, have osteoporosis,. It and it is supposed that the number will increase to 12 million and 34. Thirty-four million patients older than 50 have decreased bone density and considered high risk for osteoporosis development. Oral BPs arewere prescribed to more than 70% of the patients diagnosed with osteoporosis in US during the USA in 2003- to 2006.((American association of Oral and Maxillofacial Surgeons)). More than 190 million prescriptions arewere issued in North America. In 2003 Alendronate, alendronate is 19th most prescribed medicine (17 million prescriptions) Risedronate), risedro‐ nate is 72nd (6 million prescriptions) zolendronate), while zoledronate is used from more than

Bisphosphonate-associated osteonecrosis of the jaws is a result of treatment with BPs, bone metabolism disorders and physiological micro damagemicrodamage of the jaws interferin‐ ginterfere their biomechanical properties. Oral cavity conditions, microtrauma, infections increase the efforts of the organism to bone recovery, exceeding the ability of the hipodynamic bone. Compared to all other bones maxilla and mandible are remodeled most frequently in the human body. This is the reason why BONJ is observed only in jaw-bones. Unlike to other bones in the human body, they are not enough protected enough. It is an important fact that only a thin mucosa and a periosteum are the only barriers protecting bone from injuries. From the other side teeth are prerequisite to easy microorganisms penetration and development of internal infections–caries and periodontal disease complications. It is interesting to mention that local factors like partial and full removable dentures are also factors that increase the risk

of osteonecrosis. Smoking and continuous corticosteroid therapy are also risk factors.

According to Denn et al., all osteonecrosis cases describe todescribed in 2006 arewere associ‐ ated with nitrogen containing bisphosphonates. In scientific literature, there is no single opinion which bisphosphonates exactly leads to more frequent BONJ development. The

300,000 sick people.

**5. Pathophisiology of BONJ**

132 Immunopathology and Immunomodulation

In literature, there is no solidarity concerning bisphosphonate therapy suspension after the appearance of jaw-osteonecrosis. Marx suggests bisphosphonate therapy in onco-patients to be discussed with an oncologist in order to define positive effect/risk ratio, having in mind bisphosphonatebisphosphonate's half-life (10 years).

In literature thereThere is prevalence of BONJ with prior dental procedures compared to the so called spontaneous osteonecrosis and 33%–86% of the BONJ in literature are described after different dental procedures.

Prevailing opinion is that with ageas one gets older the danger from bisphosphonate osteo‐ necrosis increases withby 9%.

Behavior inof diagnosed osteonecrosis patients is dependent on the disease stage. It should be mentioned that American Association of Oral and Maxillofacial Surgeons proposeproposed a change in the disease nomenclature formfrom BONJ to Medicine medication-related osteo‐ necrosis of the jaws (MRONJ). The reason is an increasing number of patients with osteonec‐ rosis of the jaws have been diagnosed after other drug intake (denosumab). Due to this reason Hence, the AAOMS changeschanged the name, which was accepted in 2009, on. On their consensus conference in 2014, with a Position Paper and in addition to the new name accepts, they accepted new diagnostic and treatment strategies corresponding to the contemporary researches., as follows:

According to AAOMSAAOMS's Position Paper, it is possible to think about MRONJ if several characteristics are present:

It should be mentioned that, according to the AAOMS, risk for osteonecrosis development in patients with bone tumors treated with zoledronate is 0% to 1%. It is similar in patients treated with denozumab.denosumab. A bit lower is the valuepercentage risk in patients treated with antiangiognetic drugs, like bevacizumab –, which is 0.2%. The risk for patients with osteopo‐ rosis treated with denozumabdenosumab and zoledoronate is 0.04%. It should be noted that with increasingthe longer the period of time for treatment is increasingthe higher the risk for jaw osteonecrosis form, from 0.5% for one year of treatment to 1.1% for three year years of treatment. After all it is a matter ofThese are based on retrospective studies of treated patients, which who have not been treated surgically in the oral cavity but has develophave developed spontaneous osteonecrosis. As aA trigger factor could be underlined tooth or even multiple teeth extraction and placement of dental implants. In differentDifferent retrospective studies are shown showed that 1.6% to 14.8% of patients treated i.v.through IV with BPs for longer than a year period of time and have developed osteonecrosis developed after tooth extraction. According to a research done by Carlos Pigrau – –Serralach et al since 2014, BONJ reachescases have reached 30.2% Viridance since 2014. Viridans group Streptococcistreptococci are isolated in 83.3% formfrom the cases and AcctinomycesActinomyces spp in 39.0% from all bone histological samples.

According to other studies, between 52% and 61% from all cases described of osteonecrosis of patientscases treated with bisphosphonates are triggered after surgical treatment – –a per‐ centage that is extremely high. Retrospective research done by Kunchur R,., Need A,., Hughes T,., et al. in 2009, says stated that from0.5% out of 194 patients treated with oral bisphospho‐ nates have developed osteonecrosis occurrence is 0.5% after tooth extraction. They assumeas‐ sumed that dental implant placement iswas with similar biological load as tooth extraction. Moreover, Donggeol Lee reportsreported that 77 patients treated with bisphosphonate undergounderwent implantology treatment, with 78 placed implants with 97.4% success equal to success in normal conditions. Sebastien Hoefert and Harald Eufinger report i.v.reported IV and per os antibiotic treatment in bisphosphonate treated patients before surgical treatment.

In 2009, P. Pechalova, A. Bakardjiev, B. Vladimirov, E. Poryazova, I. Angelova, and A. Jeleva publishpublished a case of a patient with bisphosphonate osteonecrosis of the mandible of a patient after consistent oral intake of 14 month consistent intake for fourteen months of FosamaxR (alendronate) (70mg per week dosage, taken once a week) and BonvivaR (ibadro‐ nate) (150mg month dosage, taken once a month) on the occasion ofdue to severe osteoporosis with pathological fractures of vertebras. The oddly think in the case published is bone sequester covered entirely with mucosa. Without fistula..

A group formfrom ITI (Bornstein, Cionca, Momblli)), based on a vast literature in 2009 differentiate, differentiated the surgeon behavior in patients with oral and i.v.IV bisphospho‐ nate intake. They assumeassumed that, in addition to the way of intake there is, the duration of intake played an important role of the duration.. They saysaid that patients with osteopo‐ rosis/osteopenia take much lower dosage of bisphosphonate compared to cancer patients and, thus, the accumulation of the medicine should be several years. This is enough to conclude that In conclusion, patients who underwent oral surgical procedures are withhave significantly lower risk of necrosis in these patientscompared to those who had IV bisphosphonate intake.

Similar conclusions are were made by our team on the basis of 3 treatedthree patients in 3 yearswho underwent treatment for the period of three years. One of the patients were onhad an oral intake of bisphosphonates, one i.v.had IV, and one on DenozumabeDenosumabe (subcutaneоus) As part of our). Our entire biological concept treatment include includes preliminary antibiotic treatment, irrigation with chlorxeidine solutions, general and local ozone therapy, hyperbaric chamber, PRGF usage, Er;Yag:YAG laser, photodynamic therapy, and minimally invasive surgical techniques.

### **7. Conclusions**

**1.** I.v.IV bisphosphonates for treatment of primary bone tumors and bone metastasis,

**2.** Oral bisphosphonates mainly for osteoporosis and osteopenia treatment, as well as

**3.** DenozumabDenosumab–RANK ligand inhibitor–it inhibits bone resorption by inhibition of the function of osteoclasts and as an advantage could be mentioned that it is not connected to the bone so within 6 months after treatment the risk of complications is

**4.** Antiangiogenetic medications–it is used in different tumor conditions and is useful due to the fact that preventsto prevent formation of new blood vessels. It connects to signal molecules responsible for blood vessels formation and interfereinterferes the angiogenesis

According to AAOMSAAOMS's Position Paper, it is possible to think about MRONJ if several

**1.** Antiresorptive or atiangiogenicantiangiogenic treatment conducted before or in the past.,

**2.** Bone that is exposed in the maxillofacial region through intraoral or extraoral fistula(e)

It should be mentioned that, according to the AAOMS, risk for osteonecrosis development in patients with bone tumors treated with zoledronate is 0% to 1%. It is similar in patients treated with denozumab.denosumab. A bit lower is the valuepercentage risk in patients treated with antiangiognetic drugs, like bevacizumab –, which is 0.2%. The risk for patients with osteopo‐ rosis treated with denozumabdenosumab and zoledoronate is 0.04%. It should be noted that with increasingthe longer the period of time for treatment is increasingthe higher the risk for jaw osteonecrosis form, from 0.5% for one year of treatment to 1.1% for three year years of treatment. After all it is a matter ofThese are based on retrospective studies of treated patients, which who have not been treated surgically in the oral cavity but has develophave developed spontaneous osteonecrosis. As aA trigger factor could be underlined tooth or even multiple teeth extraction and placement of dental implants. In differentDifferent retrospective studies are shown showed that 1.6% to 14.8% of patients treated i.v.through IV with BPs for longer than a year period of time and have developed osteonecrosis developed after tooth extraction. According to a research done by Carlos Pigrau – –Serralach et al since 2014, BONJ reachescases have reached 30.2% Viridance since 2014. Viridans group Streptococcistreptococci are isolated in 83.3% formfrom the cases and AcctinomycesActinomyces spp in 39.0% from all bone

According to other studies, between 52% and 61% from all cases described of osteonecrosis of patientscases treated with bisphosphonates are triggered after surgical treatment – –a per‐ centage that is extremely high. Retrospective research done by Kunchur R,., Need A,., Hughes T,., et al. in 2009, says stated that from0.5% out of 194 patients treated with oral bisphospho‐

**3.** Lack of radiation therapy in the oro-facial region or metastatic disease to the jaws.

multiple myeloma, and severe osteoporosis but with lower dosage.

PagetPaget's disease and osteogenesis imperfectimperfecta.

slightly noticeable.

134 Immunopathology and Immunomodulation

signaling cascade.

characteristics are present:

histological samples.

persisting for more than 8 weeks, and

During an surgical, respectively a surgery, such as implantology treatment, especially in bisphosphonate treated patients it is vital antibiotics preparation and is vital for the primary healing process to be guaranteed, especially in bisphosphonate treated patients. Usage of Clindamycin as osteotopicosteotropic antibiotics is a gold standard assuring lackto ensure absence of post-operation infection and prevention of complications. LackThe absence of dehiscence assureensures us smooth osteointegration process and lack of bone lose. That is whyFor this purpose, the common requirements for soft tissue management, include suitable implantology system, proper operation technique, atraumatic preparation, and last but not least proper suturing materials and technique. Implantology has extra sources like PRP, PRF,, PRGF, which could be applied with great success if it's needed. The high concentration of platelets rich in growth factors (PRGF) in the operation field contributecontributes to a light and with lack of troublestrouble-free post-operative process, guaranteeing perfect conditions for the osteointegration process. In cases of post-operative complications MethronidazoleMe‐ tronidazole is a matter of choice.

#### **8. Clinical cases**

#### **8.1. Case 1**

The patient has arrived at our clinic for the first time in 2009 with pain syndrome, periodontal abscess in the 47, 44, 31, 32, 41, and 42 area due to periodontitis developed formfrom the plaque with periodontal pockets form 6from 6mm to 8mm.,combined with chronic occlusal trauma from iatrogenic sharp edges of metal-ceramic constructions made years ago (X-ray, Fig. 1). Based on the anamnesis, a treatment with medicines was found–6 months treatment with Bonviva medicine–3mg once a month parenteral, associated with diagnosed osteopenia in 2007 with quantitative osteometry over tibia bone with T-score 2,9. It was considered that due to the short-term treatment with the bisphosphonate medicine and the 5five-year period after it by keepingto keep certain protocol for preparation and implantation, an implantology treatment could be initiated. A multiple extraction of 47, 44, 42, 41, 31, and 32 was undertaken after antibiotic protection, initial ultrasonic and sand-blasting therapy divided in tointo several appointments, high-intensity and low intensity laser therapy, local and general ozone therapy and hyperbaric therapy. Extraction sockets were filled with PRGF and sutured with PTFE. As an additional prophylaxis measure from bisphosphonates induced osteonecrosis, photody‐ namic therapy was used. The healing process was without any complications.

The treatment plane includesplan included the entire sanitation of the mouth, periodontal and endodontic preparation, and placement of 11 pieces titanium screwed implants. After uncov‐ ering of the implants, tooth 43 was planplanned for extraction despite the exact root-canal treatment, due theto long term prognosis and high risk of root fracture. TillUntil that time, this particular tooth was used for temporary fixed construction abutment. Because of the short clinical crowns, crown-lengthening was planplanned and performed with Er:YAG laser, based on the preliminary analysis of the size and shape of future prosthetic crowns and attached gingiva available. X-ray surgical guide was made, along with a prototype of the future prosthetic constructions, and was used for the CT. Due to the fact of previous bisphosphonate usage, a certain surgical protocol was used again including: antibiotic prophylaxis, highintensity and low intensity laser therapy, local and general ozone therapy, hyperbaric therapy, PRGF, precise soft-tissue management and photodynamic therapy. One implant was placed in 15 area. Due to the lack of complications it was proceeded to, the placement of the rest of the planned implants proceeded. Bone type was D4 for the maxilla and D3 for the mandible. Bone spreaders were used for bone condensation. The primary stability of the implants was 15 up to 30 N/cm. Osteotomic implant beds were shaped by the usage of the existing surgical guide. Existing bone doesdid not demandeddemand additional augmentative procedures. Suture material was 4/0 PTFE. On the both jaws, fixed long-term temporary constructions waswere provided. After longer than usual period of waiting (more than ana year)), implants were uncovered by half-thickness flaps moved apically. All 11 implants were osteointegrated. After open-tray, impression technique with transfers zirconium abutments with titanium base were made. CAD-CAM zirconium constructions were madmade directly on the same model. In the frontal area constructions were made by blend-ceramic and distally full anatomy zirconium.

Complete functional rehabilitation of the masticatory apparatus was achieved. Aesthetic demands of the patient was covered completely.

**Figure 6.** Basic X-ray

platelets rich in growth factors (PRGF) in the operation field contributecontributes to a light and with lack of troublestrouble-free post-operative process, guaranteeing perfect conditions for the osteointegration process. In cases of post-operative complications MethronidazoleMe‐

The patient has arrived at our clinic for the first time in 2009 with pain syndrome, periodontal abscess in the 47, 44, 31, 32, 41, and 42 area due to periodontitis developed formfrom the plaque with periodontal pockets form 6from 6mm to 8mm.,combined with chronic occlusal trauma from iatrogenic sharp edges of metal-ceramic constructions made years ago (X-ray, Fig. 1). Based on the anamnesis, a treatment with medicines was found–6 months treatment with Bonviva medicine–3mg once a month parenteral, associated with diagnosed osteopenia in 2007 with quantitative osteometry over tibia bone with T-score 2,9. It was considered that due to the short-term treatment with the bisphosphonate medicine and the 5five-year period after it by keepingto keep certain protocol for preparation and implantation, an implantology treatment could be initiated. A multiple extraction of 47, 44, 42, 41, 31, and 32 was undertaken after antibiotic protection, initial ultrasonic and sand-blasting therapy divided in tointo several appointments, high-intensity and low intensity laser therapy, local and general ozone therapy and hyperbaric therapy. Extraction sockets were filled with PRGF and sutured with PTFE. As an additional prophylaxis measure from bisphosphonates induced osteonecrosis, photody‐

namic therapy was used. The healing process was without any complications.

The treatment plane includesplan included the entire sanitation of the mouth, periodontal and endodontic preparation, and placement of 11 pieces titanium screwed implants. After uncov‐ ering of the implants, tooth 43 was planplanned for extraction despite the exact root-canal treatment, due theto long term prognosis and high risk of root fracture. TillUntil that time, this particular tooth was used for temporary fixed construction abutment. Because of the short clinical crowns, crown-lengthening was planplanned and performed with Er:YAG laser, based on the preliminary analysis of the size and shape of future prosthetic crowns and attached gingiva available. X-ray surgical guide was made, along with a prototype of the future prosthetic constructions, and was used for the CT. Due to the fact of previous bisphosphonate usage, a certain surgical protocol was used again including: antibiotic prophylaxis, highintensity and low intensity laser therapy, local and general ozone therapy, hyperbaric therapy, PRGF, precise soft-tissue management and photodynamic therapy. One implant was placed in 15 area. Due to the lack of complications it was proceeded to, the placement of the rest of the planned implants proceeded. Bone type was D4 for the maxilla and D3 for the mandible. Bone spreaders were used for bone condensation. The primary stability of the implants was 15 up to 30 N/cm. Osteotomic implant beds were shaped by the usage of the existing surgical guide. Existing bone doesdid not demandeddemand additional augmentative procedures. Suture material was 4/0 PTFE. On the both jaws, fixed long-term temporary constructions

tronidazole is a matter of choice.

136 Immunopathology and Immunomodulation

**8. Clinical cases**

**8.1. Case 1**

**Figure 7.** Intraoral situation after preliminary treatment

**Figure 8.** X-ray- before implant insertion

**Figure 9.** Minimal invasive insertion of the first implant

After preliminary treatment, the patient receivereceived one implnantimplant in the area of the second upper right premolar (15) accompanied with clindamycin prophylaxis, PRGF activation of the implant surface and covered by PRFG fibrin membrane, combined with postimplantationpost implantation photodynamic therapy using PhotoSan® for 4 minutes, two timetimes a day tilluntil the suture removal, and ozone therapy in the first three days combined with hyperbaric-chambers.

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**Figure 10.** Implant insertion in second quadrant

**Figure 8.** X-ray- before implant insertion

138 Immunopathology and Immunomodulation

**Figure 9.** Minimal invasive insertion of the first implant

combined with hyperbaric-chambers.

After preliminary treatment, the patient receivereceived one implnantimplant in the area of the second upper right premolar (15) accompanied with clindamycin prophylaxis, PRGF activation of the implant surface and covered by PRFG fibrin membrane, combined with postimplantationpost implantation photodynamic therapy using PhotoSan® for 4 minutes, two timetimes a day tilluntil the suture removal, and ozone therapy in the first three days

**Figure 11.** Implant insertion in lower jowjaw

**Figure 12.** Activation of implant surface with PRGF

**Figure 13.** PRGF membrane for covering of the implants

The high concentration of platelets rich in growth factors (PRGF) in the operation field contributecontributes to a light and with lack of troubles trouble-free post-operative process, guaranteeing perfect conditions for the osteointegration process.

The process of tissue repair iswas based on a complex cascade of biological events controlled by a long list of biologically active growth factors and proteins. The spatial and temporal action of this family of mediators on the tissue-damaged area regulates the mechanisms and phases that govern tissue repair and regeneration

**Figure 14.** Pure ozone injected subcutaneously in the manipulation area

The high concentration of platelets rich in growth factors (PRGF) in the operation field contributecontributes to a light and with lack of troubles trouble-free post-operative process,

The process of tissue repair iswas based on a complex cascade of biological events controlled by a long list of biologically active growth factors and proteins. The spatial and temporal action of this family of mediators on the tissue-damaged area regulates the mechanisms and phases

guaranteeing perfect conditions for the osteointegration process.

that govern tissue repair and regeneration

**Figure 13.** PRGF membrane for covering of the implants

**Figure 12.** Activation of implant surface with PRGF

140 Immunopathology and Immunomodulation

Another method used in our biological concept is ozone therapy. There are several known actions of ozone onin the human body, such as anti-microbial, immunostimulating, antihi‐ poxyc, analgesic, detoxicating, bioenergetics and biosynthetic (activation of the metabolism of carbohydrates, proteins and lipids)), etc. Ozone is a relatively rare and unstable molecule composed of three oxygen atoms O3. The normal oxygen molecule has two oxygen atoms O2. Stratospheric ozone is important in the earth system because it absorbs ultraviolet radiation from the sun, protecting life on earth. The beneficial biological effects of ozone, is its antimicrobialantimicrobial activity. How we can derive ozon?the ozone? Using a simple electric arc, we can produce ozone. In case, for example, of bone regeneration, for example, the purpose of ozone using ozone is the initial local growth factor expression and stimulatetion stimulate the migration of osteoprogenitor cells to the wound site and subsequentlyand subsequently, in a controlled fashion, to direct their differentiation to the osteogenic cell line. Throughout this process, another set of factors will regulate the dynamic equilibrium between cell inhibi‐ tion and proliferation, as well as angiogenesis and extracellular matrix formation. Ozonegenerator can introduce pure ozone in saline. Ozone infusion is also precious for the fact that it generates a therapeutic effect on faster post op wound healing and preliminary preparation of the patient by avoiding further complications and assuring on 99% smooth primary closure. Anti-microbial effect of ozone asis a result of its action on cells by damaging its cytoplasmatic membrane due to ozonolysis of dual bonds and also ozone-induced modification of intercel‐ lular contents because of secondary oxidants effects. This action is non-specific and selective to microbial cells, it does not dameagedamage human cells because of their major antioxidative ability. Ozone is very efficient in antibiotics resistant strains. Its anti-microbial activity increases in liquid environment of acidic pH. In viral infections, the ozone action lies in the intolerance of infected cells to peroxides and change of activity of reverse transcriptase, which takes part in synthesis of viral proteins.

**Figure 15.** Usage of photodynamic therapy contributecontributes to cell destruction of the microorganisms and pre‐ ventprevents complications and accelerateaccelerates wound healing

**Figure 16.** Photodynamic therapy with photodynamic intraoral tray

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**Figure 17.** Minimal invasive extraction of 43- root separation

intolerance of infected cells to peroxides and change of activity of reverse transcriptase, which

**Figure 15.** Usage of photodynamic therapy contributecontributes to cell destruction of the microorganisms and pre‐

ventprevents complications and accelerateaccelerates wound healing

**Figure 16.** Photodynamic therapy with photodynamic intraoral tray

takes part in synthesis of viral proteins.

142 Immunopathology and Immunomodulation

**Figure 18.** Minimal invasive extraction of 43

**Figure 19.** Granulation tissue ablation with Er:YagYAG laser

**Figure 20.** Palatal connective tissue graft

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**Figure 21.** Palatal connective tissue graft in PRGF

**Figure 19.** Granulation tissue ablation with Er:YagYAG laser

144 Immunopathology and Immunomodulation

**Figure 20.** Palatal connective tissue graft

**Figure 22.** Bone graft material mixed with PRGF

**Figure 23.** Bone graft material mixed with PRGF in postextractionpost-extraction socket

**Figure 24.** PRGF-clot cloth

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**Figure 25.** Covering of bone graft material with the PRGF clotcloth

**Figure 23.** Bone graft material mixed with PRGF in postextractionpost-extraction socket

**Figure 24.** PRGF-clot cloth

146 Immunopathology and Immunomodulation

**Figure 26.** Suturing of the palatal connective tissue graft over the postextracted socetpost-extracted socket

**Figure 27.** Soft healing process

**Figure 28.** The end result

**Figure 29.** End result after treatment

#### **8.2. Case 2**

**Figure 27.** Soft healing process

148 Immunopathology and Immunomodulation

**Figure 28.** The end result

**Figure 29.** End result after treatment

A female patient, 57 years old, S. H. in good general good health, non-smoker, was received at the clinic with complainscomplaints of mobility of the entire upper jaw prosthetic fixed construction, accompanied with pain and inability to eat, dating back to 3 months earlier. Clinical exam findfound out significant mobility (4th degree by Miller) of metal-ceramic fixed one-piece bridge prosthetic construction of the upper jaw. (Fig. 2). On the X-ray (Fig.1), 4 screwing dental implants arewere observed, "Tramonte design" and 4four residual roots were in terminal stage. There iswas a significant bone resorption around the implants visible on the x-ray (proven lack of osteointegration.). From the patient histori ispatient's history, it was known that these implants arewere placed 5five years ago. AnA year later, patient beginsbegan treatment with Fozamax per os once a week withfor the duration 2of two years because of osteoporosis diagnosediagnosis (T-scorscore-2.9%).

**Figure 30.** Initial X-ray

**Figure 31.** Intraoral view

After initial periodontal therapy, with ultrasonic device and highintensityhigh intensity laser, under antibiotic cover (clindamycin 600mg. /./8h per os), 2two times hyperbaric chamber before and three times after the dental procedures, saline enriched with ozone 9nine times and three were locally applied subcutaneously, photodynamic therapy tilluntil the 10th den after the dental procedure of removing all residual roots and implants. In the extraction sockets were placed PRGF cloth, collagen sponge and the wound were sutured. Post-operative period was smooth without any pain or complications. The patientspatient was given an immediate fabricated total removable dentures.

**Figure 32.** Intraoral view after bridge removal

**Figure 33.** All implants, teeth and bridge after removal

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**Figure 34.** Granulation tissue ablation with Er:YagYAG laser

before and three times after the dental procedures, saline enriched with ozone 9nine times and three were locally applied subcutaneously, photodynamic therapy tilluntil the 10th den after the dental procedure of removing all residual roots and implants. In the extraction sockets were placed PRGF cloth, collagen sponge and the wound were sutured. Post-operative period was smooth without any pain or complications. The patientspatient was given an immediate

fabricated total removable dentures.

150 Immunopathology and Immunomodulation

**Figure 32.** Intraoral view after bridge removal

**Figure 33.** All implants, teeth and bridge after removal

**Figure 35.** PRGF – cloth

**Figure 36.** Covering with collagen sponge and suturing

**Figure 37.** Day after surgery

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**Figure 38.** Ten days after surgery

**Figure 36.** Covering with collagen sponge and suturing

152 Immunopathology and Immunomodulation

**Figure 37.** Day after surgery

**Figure 39.** Three weeks after surgery

**Figure 40.** Intraoral view after implant insertion

**Figure 41.** Intraoral view after suturing

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**Figure 42.** Two weeks after surgery

**Figure 40.** Intraoral view after implant insertion

154 Immunopathology and Immunomodulation

**Figure 41.** Intraoral view after suturing

**Figure 43.** Panoramic X-ray after surgery

**Figure 44.** Four weeks after surgery

#### **8.3. Case 3**

A female patient, 54 years old, in good general health condition, smoker (over 10 cigarettes per day), was received at the clinic with complainscomplaints from fixed prosthetic construc‐ tion mobility on the upper jaw, poor aesthetics, and inability to eat normally. X-ray proves anand aggressive periodntoalperiodontal disease with more than 9-10mm910mm bone resorption of interdental bone. PatientPatient's history sayssaid that she is was on Denosu‐ mabdenosumab (subcutaneously every 6 months) during for the last 3three years –in treatment for osteoporosis diagnose. The treatment plan was similar with case N2 and the healing process went smoothsmoothly.

**Figure 45.** Intraoral view

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**Figure 46.** Postextracted sockets

**Figure 44.** Four weeks after surgery

156 Immunopathology and Immunomodulation

went smoothsmoothly.

**Figure 45.** Intraoral view

A female patient, 54 years old, in good general health condition, smoker (over 10 cigarettes per day), was received at the clinic with complainscomplaints from fixed prosthetic construc‐ tion mobility on the upper jaw, poor aesthetics, and inability to eat normally. X-ray proves anand aggressive periodntoalperiodontal disease with more than 9-10mm910mm bone resorption of interdental bone. PatientPatient's history sayssaid that she is was on Denosu‐ mabdenosumab (subcutaneously every 6 months) during for the last 3three years –in treatment for osteoporosis diagnose. The treatment plan was similar with case N2 and the healing process

**8.3. Case 3**

**Figure 47.** The extracted teeth

**Figure 48.** Granulation tissue ablation with Er:YagYAG laser

#### **Figure 49.** PRGF clotcloth

**Figure 50.** Covering with collagen sponge and suturing

**Figure 51.** The immediate denture

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#### **Figure 52.** Three weeks after extraction

**Figure 49.** PRGF clotcloth

158 Immunopathology and Immunomodulation

**Figure 50.** Covering with collagen sponge and suturing

**Figure 51.** The immediate denture

\*All case-pictures are Dr. Abadzhiev's private practice patiens.

**Table 1.** Usage and dosage of the bisphosphonates

After R.E. Marx

**Table 2.** Staging and Treatment Strategies

Position Paper of American Association of Oral and Maxillofacial Surgeons (2014)

#### **Author details**

Metodi Abadzhiev\*

Address all correspondence to<EMAIL_ADDRESS>
MU-Varna, Faculty of Dental Medicine, Bulgaria


*3.4. Agronomic Relevance*

In a fruit orchard, the canopy dimension dynamically changes, and, consequently, the spectral distribution of the incoming radiation varies widely, as the light penetrates and scatters within the tree canopy due to the structure and optical properties of plant organs [65,66]. In general, the spectral modifications of light inside the tree canopy have a crucial role in growth partitioning among fruit and shoots, affecting the allocation to developing fruits in plant growth and fruit quality [67]. The effects of modification of the CRYs and PHYs abundance and photosensitivity of plants in response to the changing light on cross talks during host-pathogen interaction remain to be studied in fruit trees,

and the molecular mechanisms underlying the interaction of monochromatic light with plant and bacteria remain poorly understood because they are influenced by environmental conditions. Results obtained in experiments in vitro, with pure cultures of *E. amylovora* Ea273 strain (Figure 1), and in vivo, with infected transgenic *Dar Gazi* lines (Figure 12), clearly indicated that the quality of the light and the photoreceptor-mediated signals affect the growth of the pathogen and its infectivity and aggressiveness. In this respect, the use of the LED technology can be valuable to develop new procedures for sustainable and non-invasive control of this pathogen.

These findings also have great economic importance because *PR1* is used as a lookout pathogen presence. During the period of fruit conservation in dark conditions, an interruption of these light conditions through BL flesh could repress the insurgence, the development, and bacterial proliferation. Even if there are not many studies on *PR*s and woody fruit crop plants, it has recently been presented that genetically engineered phytochrome A cherry plants showed the highest level of tolerance to *Pseudomonas syringe* pv *mors-prunorum*, when compared to the wild type plants [48].

Finally, one of the four plant food allergens, the Bet v 1 superfamily, contains ten pathogenesis-related proteins [68]. Our findings could be further explored to study the regulation of this allergen-related protein and the relative reduction of its presence and accumulation by modulating the lighting during the post-harvest fruit conservation.