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## **Nitric Oxide: Key Features in Spermatozoa**

Florentin-Daniel Staicu and Carmen Matas Parra

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67551

#### **Abstract**

Several *in vitro* studies have pointed to the importance of nitric oxide (NO) in the female and male reproductive system in mammals. Its functions vary from preventing oocyte aging, improving the integrity of the microtubular spindle apparatus in aged oocytes, modulating the contraction of the oviduct, to regulating sperm physiology by affecting the motility, inducing chemotaxis in spermatozoa, regulating tyrosine phosphorylation, enhancing the sperm-zona pellucida binding ability, and modulating the acrosomal reaction. In spermatozoa, NO exerts its functions in different ways, which involve key elements such as the soluble isoform of guanylate cyclase, cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), adenylate cyclase, and the extracellular signal-regulated kinase (ERK) pathway. Furthermore, NO leads to the S-nitrosylation of several sperm proteins, among them a substantial group associated with energy generation and cell movement, but also with signal transduction, suggesting a role for S-nitrosylation in sperm motility and in modulating the sperm function, respectively. In this chapter, an overview of how NO modulates the sperm physiology is presented, based on the knowledge acquired to this day.

**Keywords:** nitric oxide, nitric oxide synthase, S-nitrosylation, spermatozoa, fertilization

#### **1. Introduction**

NO is a small hydrophobic molecule which can easily diffuse through biological membranes [1]. *In vivo*, it is synthesized during the conversion of L-arginine to L-citrulline by nitric oxide synthase, with the help of co-factors such as the reduced form of nicotinamide adenine dinucleotide phosphate, flavin mononucleotide, flavin adenine dinucleotide, and tetrahydrobiopterin [2].

© 2017 The Author(s). Licensee InTech. 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.

The nitric oxide synthase (NOS) may be found in three different isoforms. Two of them, the endothelial and the neuronal NOS (eNOS, nNOS), require calcium/calmodulin to be activated and are responsible for the continuous basal release of NO. The third isoform, known as inducible NOS (iNOS), is calcium independent [3, 4]. Since NOS activity depends on the availability of its substrate and its co-factors, all these elements jointly determine the cellular rates of NO synthesis [5].

Substantial evidence indicates that NO is a crucial biological messenger involved in a wide variety of physiological and pathological processes in different systems in mammals, including the vascular, nervous, and reproductive system [1, 6].

### **2. NOS/NO duo in the reproductive system**

The NOS/NO duo regulates key functions in both the female and male reproductive systems [6].

All three isoforms of NOS have been identified in the oviduct [7, 8], oocytes, and cumulus and corona cells [9, 10] of several species [7, 11, 12]. The expression of NOS isoforms differs during the estrous cycle in the follicles as well as in the oviduct [13]. Tao *et al*. [10] showed that the immunoreactivity of eNOS in early antral follicles was restricted to the oocyte and increased from small and medium to large follicle-enclosed oocytes. In contrast, no immunoreactivity for iNOS was found in primordial, early antral follicle, or the cumulus-oocyte complexes aspirated from small and medium follicles.

In the oviduct, the endogenous basal release of NO regulates its contraction and the ciliary beating of the ciliated epithelial cells and induces chemotaxis in human spermatozoa via activation of the nitric oxide/soluble isoform of guanylate cyclase/cyclic guanosine monophosphate pathway (NO/sGC/cGMP) [14–16]. NO forms a vital component of the oocyte microenvironment and has been positively implicated in meiotic resumption [17], in preventing oocyte aging and improving the integrity of the microtubular spindle apparatus in aged oocytes [18]. It may also contribute as an anti-platelet agent during implantation [19].

As far as the male gamete is concerned, research was first concentrated on determining the effects of NO-releasing compounds on sperm motility and viability. Low concentrations of sodium nitroprusside (SNP), an NO-releasing compound, stimulated sperm hyperactivation in mouse, fish, and hamster [20–22] and were beneficial to the maintenance of post-thaw human sperm motility [23]. On the other hand, high concentrations of NO-releasing compounds decreased sperm motility [20, 24–26].

Numerous studies have also been conducted to determine the presence and localization of NOS in sperm from several species (**Table 1**). For example, Herrero *et al*. [27] located nNOS in the head of freshly ejaculated human spermatozoa, with a more concentrated fluorescent staining toward the equatorial region. O'Bryan *et al*. [28] described the pattern of eNOS expression in human spermatozoa, finding that morphologically normal spermatozoa exhibited post-acrosomal and equatorial eNOS immunostaining. Interestingly, though, abnormally shaped sperm cells exhibited aberrant staining, especially in the midpiece and/or head region, which correlated negatively with the percentage of motile sperm.

**Table 1.** Summary of *in vitro* studies and the techniques used to identify NOS isoforms in different species.

NOSs were revealed in mature mouse spermatozoa by means of biochemical techniques and Western blot. Herrero *et al*. [29] showed that mouse spermatozoa can synthesize L-citrulline, depending on the concentration of L-arginine present in the incubation medium while different concentrations of N(G)-nitro-L-arginine methyl ester (L-NAME) inhibit the formation of the amino acid. Furthermore, when sperm protein extracts were incubated under denaturing and nonreducing conditions and then subjected to immunoblotting assay, a protein fraction of 140 kDa was recognized by the three anti-NOS antibodies.

Bull spermatozoa were examined for the presence of constitutive NOS [30]. NO generation seemed to be enhanced by L-arginine and abolished by the NOS-inhibitor, L-NAME. In addition, Meiser and Schulz [30] verified the presence of NOS in bull sperm cells by immunohistochemistry, which was confirmed by Western blot. Confocal laser microscopy localized nNOS-related immunofluorescence at the acrosome cap and the main part of the flagellum. The same technique also identified eNOS staining spread over the spermatozoan head. Moreover, when these findings were confirmed by Western blot, immunoreactive bands at 161 kDa (nNOS) and 133 kDa (eNOS) were identified.

Hou *et al*. [31] investigated whether boar sperm can generate NO, finding that porcine spermatozoa synthesized low levels of NO under noncapacitating conditions, but that the NO concentration almost doubled when sperms were capacitated. Furthermore, NO production was significantly inhibited when capacitated sperms were treated with L-NAME. In another study [32], Western blot analysis was performed to identify NOS enzymes in boar sperm samples. The immunoblots showed three distinct bands: ~160, ~130, and ~135 kDa, corresponding to nNOS, iNOS, and eNOS, respectively.

NO production was evaluated in stallion spermatozoa before and after freezing/thawing [33] by means of flow cytometry, after loading the sperm suspension with an NO detection probe. NO synthesis was positively correlated with sperm motility after thawing and, interestingly, the presence of egg yolk in the semen extender radically reduced the amount of NO produced. The authors further investigated in fresh and frozen/thawed stallion sperm the presence of NOS enzymes by Western blot, using anti-nNOS, anti-eNOS, and anti-universal NOS antibodies. Two bands of approximately 83 kDa and 96 kDa were labeled by the antibodies anti-nNOS and anti-eNOS, respectively. Moreover, the other antibody, which recognized an epitope present in all the NOS isoforms described so far, showed two similar bands of 84 and 92 kDa.

Recently, Liman and Alan [34] investigated the localization of NOS isoforms in spermatozoa within the intratesticular and excurrent duct systems of adult domestic cats. Overall, the spermatozoa head did not exhibit immunoreactivity. On the other hand, immunoreactivity for all three isoforms was observed in the flagellum, in the proximal cytoplasmic droplets of spermatozoa (located in the neck region) within the lumen of the intratesticular and efferent ducts, in the epididymal duct of the caput epididymis, and in the distal cytoplasmic droplets of spermatozoa (located at the mid-principal piece junction of the tail) within the lumen of corpus and cauda epididymis and the vas deferens.

#### **3. Role of NO on sperm functionality**

Several *in vitro* studies were conducted in order to determine the effects that NO has on sperm physiology (**Figure 1**). It has been shown that NO affects sperm motility [28, 35, 36], acts as chemoattractant [16, 37], regulates the tyrosine phosphorylation of different sperm proteins [38, 39], enhances the sperm-zona pellucida binding ability [40], and modulates the acrosomal reaction [41, 42].

In detail, NO seems to play an important role in the maintenance of sperm motility at physiological levels. A study [36] showed that the basal release of NO by spermatozoa from normozoospermic samples tended to be greater than that from asthenozoospermic samples, suggesting a physiological and beneficial role for endogenous NO in the preservation of sperm motility. These observations agree with a previous report that normozoospermic spermatozoa express more NOS and generate more nitrite than asthenozoospermic spermatozoa [35]. On the other hand, as previously mentioned, it has been shown that spermatozoa with an abnormal morphology show aberrant staining for eNOS, which was negatively correlated with the motility [28]. A detrimental effect on motility has also been reported by Rosselli *et al*. [24] and Weinberg *et al*. [25] when millimolar concentrations of exogenous NO donors were added to sperm samples.

**Figure 1.** Some aspects of the sperm physiology modulated by the NOS/NO system. At physiological levels, endogenous NO has a beneficial role in maintaining sperm motility, enhances tyrosine phosphorylation, which, in turn, promotes the capacitation process. NO also increases the sperm-zona pellucida binding ability and leads to a rise in the percentage of reacted spermatozoa, especially in the presence of follicular fluid or protein-enriched extracts of follicular fluid.

It has been suggested that, upon approaching and entering the cumulus oophorus, both NO and progesterone, which are synthesized by the cumulus cells [9, 10, 43–45], provide a synergistic stimulus to mobilize stored calcium in the sperm neck/midpiece [46]. As a consequence, they can modulate flagellar activity and contribute to the hyperactivation that is vital for penetration of the oocyte vestments [47].

Interestingly, it has also been suggested that NO may exert a chemoattractant effect on spermatozoa. In fact, the percentage of mouse sperm migrating toward the medium containing an NO donor increased significantly [37]. Similar results were obtained when human spermatozoa were exposed to an NO donor [16]. In the latter case, the signal transduction pathway was also studied. It was proposed that NO exerts its chemoattractant effect through the activation of the NO/sGC/cGMP pathway, since the use of an NO scavenger and/or an sGC and cGMPdependent protein kinase inhibitor reverted the NO donor-induced migration of sperm.

Since tyrosine phosphorylation in different sperm proteins is associated with the capacitation process [48], this aspect was investigated in order to further define the involvement of NO in capacitation. Herrero *et al*. [38] observed an increase in tyrosine phosphorylation when human sperm capacitation was accelerated by an NO-releasing compound. On the other hand, when sperm capacitation was inhibited by L-NAME, there was an attenuation in the tyrosine phosphorylation of sperm proteins. In addition, Thundathil *et al*. [39] reported that L-NAME prevented, and a NO donor promoted, the increase in threonine, glutamine, and tyrosine phosphorylation in human spermatozoa. Furthermore, the addition of L-arginine reversed the inhibitory effect of L-NAME on the capacitation and the associated increase in phosphorylation.

The correlation between NO and sperm-zona pellucida binding ability was investigated by Sengoku *et al*. [40], who reported that when treated with low concentrations of a NO donor, the number of spermatozoa which binds to the hemizona is higher than in sperm treated with a higher concentration. Additionally, a NO quencher lowered the enhancement of sperm binding by the NO donor.

NO also seems to modulate the acrosome reaction. The percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was studied when human spermatozoa were capacitated in the presence/absence of NO-releasing compounds or NOS inhibitors [38]. NO donors induced sperm cells to respond faster to human follicular fluid, whereas NOS inhibitors decreased the percentage of acrosome reaction. Similar results were obtained by Revelli *et al*. [41], who showed that different NO-releasing compounds were able to increase the percentage of reacted spermatozoa in the presence of protein-enriched extracts of human follicular fluid. Also, hemoglobin, a NO scavenger, inhibited the follicular fluid–induced acrosomal reaction. In an in-depth analysis of the signaling pathway of the nitric oxide–induced acrosome reaction in human spermatozoa [42], the authors suggested that the acrosome reactioninducing effect of exogenous NO on capacitated human spermatozoa is accomplished via the NO/sGC/cGMP pathway, which leads to the activation of cGMP-dependent protein kinase (PKG). In fact, both the intracellular cGMP levels and the percentage of reacted spermatozoa were significantly increased after incubation with SNP. Furthermore, the SNP-induced acrosome reaction was significantly reduced in the presence of sGC inhibitors, a reduction that was reversed by the addition of a cell-permeating cGMP analogue to the incubation medium. Finally, PKG inhibition reduced the SNP-induced acrosome reaction.

#### **4. NOS-activating molecules**

As previously stated, NOS activity depends on the availability of its substrate and co-factors [5]. However, the scientific literature does not include many studies on the molecules present in the female reproductive tract which may activate, in one way or another, NOS enzymes in spermatozoa.

Starting from follicular fluid samples, Revelli *et al*. [41] obtained a protein-enriched follicular fluid solution (PFF), which was then used to study its effects on NOS activity, citrulline synthesis, and acrosome reaction in human sperm. Interestingly, this study showed for the first time that the endogenous NOS activity of human sperm may be increased by PFF. Moreover, the authors demonstrated that PFF-mediated induction of sperm NOS activity leads to acrosome reaction in the same cells, thereby, establishing a link between follicle-derived substances, the activation of NO synthesis in sperm and biological responses.

Furthermore, the increase in NO synthesis mediated by PFF was not associated with a rise in the expression of NOS catalytic units, which is not surprising since specialized cells possess very poor, if any, transcriptional activity [41]. The authors hypothesized that PFF first determines the transient enzyme activation of sperm NOS, which is subsequently strengthened by a more stable modification of the enzyme.

However, more studies should be performed in order to identify the NOS-activating molecule(s) in the follicular fluid.

#### **5. NO pathway in spermatozoa**

In spermatozoa, NO acts via three main pathways (**Figure 2**) [13]. First, NO is able to activate sGC, leading to a rise in the intracellular levels of cGMP [49]. The latter activates the cyclic nucleotide-gated channels (CNG) localized in the flagellum of mammalian spermatozoa [50, 51]. These channels seem to play an important role in the sperm motility control, by allowing the entry of Ca2+ ions to the cytoplasm during the capacitation process of mammal sperm [50]. Their activation is one of the first events that occur during capacitation in the mouse spermatozoa [52]. cGMP also activates PKG [53, 54], which is involved in the serine/threonine phosphorylation of proteins that promote sperm capacitation and the acrosome reaction [55, 56]. Furthermore, since cGMP and cAMP compete for the catalytic sites of phosphodiesterases [57, 58], an increase in the intracytoplasmic cGMP concentration may inhibit cAMP degradation via cyclic nucleotide phosphodiesterase type 3 [59], thus increasing cAMP intracellular levels and activating protein kinase A (PKA). The latter leads to an increase in protein tyrosine phosphorylation [60].

Second, NO is directly involved in tyrosine phosphorylation by modulating the cAMP/PKA and the extracellular signal-regulated kinase (ERK) pathways. The cAMP/PKA pathway can be influenced by NO via activation of sGC (as described above), but it can also be regulated directly. In fact, S-nitrosylation of adenylate cyclase (AC) has been suggested as a possible mechanism of action of NO [61]. Low levels of NO may activate AC, consequently increasing the cAMP concentration and activating PKA [62]. However, high levels of NO can inhibit AC [61]. As far as the ERK pathway is concerned, NO reacts with the cysteine residues of the RAS protein, inducing its activation [35]. In turn, RAS triggers the RAF, MEK, and ERK1/2 complex, necessary for tyrosine phosphorylation [63].

Third, NO regulates the post-translational protein modification in spermatozoa via S-nitrosylation [64], a process similar to phosphorylation and acetylation [65, 66]. S-nitrosylation consists of the covalent incorporation of NO into thiol groups (-SH) to form S-nitrosothiols (S-NO), a modification that is selective and reversible [13].

**Figure 2.** Representation of the main pathways through which NO acts in spermatozoa. NO leads to an increase in the intracellular levels of cyclic guanosine monophosphate (cGMP) by activating the soluble isoform of guanylate cyclase (sGC). The cGMP can activate the cyclic nucleotide-gated channels (CNG) localized in the flagellum of mammalian spermatozoa, which regulate the influx of Ca2+ ions to the cytoplasm during the capacitation process and also activates the cGMP-dependent protein kinase (PKG), leading to the serine/threonine phosphorylation of different proteins. It can also inhibit cyclic adenosine monophosphate (cAMP) degradation via cyclic nucleotide phosphodiesterase (PDE), which leads to the activation of cAMP-dependent protein kinase A (PKA) and tyrosine phosphorylation. Furthermore, NO is involved in the tyrosine phosphorylation process in a direct manner, by activating adenylate cyclase (AC) and the extracellular signal-regulated kinase (ERK) pathway. Finally, NO determines post-translational protein modification in spermatozoa via S-nitrosylation.

#### **6. Function of S-nitrosoproteins in spermatozoa**

An extensive study by Lefièvre *et al*. [64] described a large number of proteins present in the sperm of normozoospermic men, which can be subjected to S-nitrosylation in the presence of NO donors. Although the function of some nitrosylated proteins remains to be discovered, a considerable group of them are known to be metabolic proteins and proteins associated with energy generation and cell movement, suggesting a role for S-nitrosylation in sperm motility. This agrees with a previous proteomic analysis [67], in which the most abundant group was also involved in energy production.

Other considerable groups of proteins were those involved in signal transduction, which agrees with a role for S-nitrosylation in modulating the sperm function [64]. Interestingly, since sperms are generally assumed to be transcriptionally inactive, a small percentage of the S-nitrosylated proteins identified by Lefièvre *et al*. [64] was related to transcription. Previous proteomic studies in sperm also observed the presence of proteins involved in transcription [67, 68]. However, when comparing the human sperm S-nitrosoproteome with proteins identified during a proteomic study of sperm-oocyte interaction, only three proteins were found in common, suggesting that S-nitrosylation is not a regulatory mechanism employed during fertilization [64, 69].

It is known that the mobilization of Ca2+ stored in the sperm neck/midpiece is necessary for the hyperactivation process [46]. The Ca2+ store in the neck of the sperm coincides with the region occupied by the redundant nuclear envelope (RNE) [70] and in order to mobilize Ca2+ from this site, ryanodine receptors (RyRs), which are intracellular Ca2+-release channels involved in regulation of cytosolic calcium levels [71], need to be activated. These proteins contain a large number of thiol groups and are thus prone to S-nitrosylation by NO [64, 72, 73]. S-nitrosylation can potentiate the opening of RyRs [74–79], probably through the generation of the membrane permanent product S-nitrosocysteine [80]. It has been shown that an increase in Ca2+ induced by NO is accompanied by an increase in S-nitrosylation levels of endogenous RyRs [81, 82] while these Ca2+ channels may be inhibited under strongly nitrosylating conditions or at high doses of NO (**Figure 3**) [76, 79, 82]. Furthermore, progesterone acts synergistically with NO to mobilize Ca2+ in the sperm neck/midpiece by activation of RyRs [47], contributing to the hyperactivation process.

**Figure 3.** S-nitrosylation process. NO acts on the thiol groups (-SH) of the cysteines in proteins to form S-nitrosothiols (S-NO). At the sperm neck/midpiece, the S-nitrosylation occurs in ryanodine receptors (RyRs) allowing the release of calcium from the redundant nuclear envelope (RNE), which is required for sperm hyperactivation. Adapted and modified from López-Úbeda and Matás [13].

Other examples of proteins which can undergo S-nitrosylation in sperm and have a known biological significance are the A-kinase anchoring proteins (AKAPs) [64]. Both AKAP3 and AKAP4 are present in the fibrous sheath of the sperm flagellum, control PKA activity and undergo phosphorylation during the capacitation process [83–85]. AKAP complexes also modulate the motility of sperm. In fact, phosphodiesterase inhibitors were seen to significantly increase sperm motility [86], whereas PKA-anchoring inhibitor peptides arrested sperm motility [87]. Since the effects of NO on sperm motility are well established, the S-nitrosylation of AKAPs would be an interesting subject for additional studies.

A number of heat shock proteins (HSPs) may also be targets of S-nitrosylation in sperm [64], and some of them have been reported to act as important modulators of sperm capacitation. For instance, Asquith *et al*. [88] reported that heat shock protein 1 and endoplasmin undergo tyrosine phosphorylation during mouse sperm capacitation, whereas Nixon *et al*. [89] suggested that they form part of a zona pellucida complex, allowing successful spermegg interaction in the same species. Heat shock 70 kDa protein 8 and heat shock protein 90α also undergo tyrosine phosphorylation during human sperm capacitation [83], but whether they function in a zona receptor complex is still unknown [64]. Furthermore, HspA2 has been shown to be a marker of sperm maturity [90], its expression in infertile men with idiopathic oligoteratozoospermia being lower than in normozoospermic men [91].

#### **7. Concluding remarks**

In recent years, our knowledge of the involvement of the NOS/NO system in mammalian fertilization has grown, and there is clear evidence that NO acts as a significant modulator of the male and female gamete. However, many aspects regarding the NOS/NO duo, such as the presence of NOS-activating molecule(s) in the fertilization site or how the biological function of the S-nitrosylated proteins changes, remain to be discovered. Shedding light on these mechanisms will increase our understanding of the etiopathology of subfertility/infertility problems and how such problems can be overcome.

#### **Acknowledgements**

This work was supported by H2020 MSC-ITN-EJD 675526 REP-BIOTECH, the Spanish Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund (FEDER), Grants AGL2015–66341-R.

#### **Author details**

Florentin-Daniel Staicu and Carmen Matas Parra\*

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Physiology, Faculty of Veterinary Science, University of Murcia, Spain

#### **References**


*Article* **Is Corticalization in Radiographs Related to a Higher Risk of Bone Loss around Dental Implants in Smoking Patients? A 5-Year Observation of Radiograph Bone-Texture Changes**

**Tomasz Wach 1,\*, Piotr Hadrowicz 2, Grzegorz Trybek 3,4, Adam Michcik 5 and Marcin Kozakiewicz 1**

**Abstract:** Background: Currently, the topic of dental implants is widely researched. However, still compromising are the factors that can affect implant loss as a consequence of marginal bone loss. One of the factors is smoking, which has a devastating effect on human health and bone structure. Oral health and jaw condition are also negatively affected by smoking. The aim of this study was to evaluate the peri-implant jawbone corticalization phenomenon in tobacco smokers. Methods: A total of 2196 samples from 768 patients with an implant in the neck area were checked, and texture features were analyzed. The corticalization phenomenon was investigated. All analyses were performed in MaZda Software. The influence of corticalization was investigated as a factor on bone structure near the implant neck. The statistical analysis included a feature distribution evaluation, mean (*t*-test) or median (W-test) comparison, analysis of regression and one-way analysis of variance or Kruskal–Wallis test as no normal distribution or between-group variance was indicated for the significant differences in the investigated groups. Detected differences or relationships were assumed to be statistically significant when *p* < 0.05. Results: The research revealed that MBL was correlated with smoking after 5 years (0.42 mm ± 1.32 mm 0 mm ± 1.25 mm), the Corticalization Index was higher in the smoker group on the day of surgery, and it became higher after 5y of observation (185.98 ± 90.8 and 243.17 ± 155.47). The implant-loss frequency was higher in the group of smokers, too, compared to non-smokers (6.74% and 2.87%). The higher the torque value during the implant placement, the higher the Corticalization Phenomenon Index. Conclusions: The research revealed a correlation between smoking and changes in bone structure in radio textures near the implants. The corticalization phenomenon is important, may be detected immediately after implant placement and may be one of the indicators of the implant success rate.

**Keywords:** bone remodeling; dental implant; smoking; torque; marginal bone loss; intraoral radiographs; radiomics; texture analysis

#### **1. Introduction**

Tobacco smoking has a devastating effect on human health and is one of the leading risks of early death. Smoking was the second risk factor for deaths in the world in 2019. Nearly 7.69 million people died because of this addiction [1–3]. There are many health disorders caused by smoking. For example, it increases the risk of many types of cancer and heart and lung diseases and increases the risk of pregnancy complications, oral cavity

**Citation:** Wach, T.; Hadrowicz, P.; Trybek, G.; Michcik, A.; Kozakiewicz, M. Is Corticalization in Radiographs Related to a Higher Risk of Bone Loss around Dental Implants in Smoking Patients? A 5-Year Observation of Radiograph Bone-Texture Changes. *J. Clin. Med.* **2023**, *12*, 5351. https:// doi.org/10.3390/jcm12165351

Academic Editors: Adam Piorkowski, Rafał Obuchowicz, Andrzej Urbanik and Michał Strzelecki

Received: 14 June 2023 Revised: 20 July 2023 Accepted: 28 July 2023 Published: 17 August 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

diseases, neuroticism (also in groups of e-cigarette smokers) and more [4–9]. Marginal bone loss is strongly connected with smoking and also with the degree of smoking [10–14]. On the other hand, it is not stated that tobacco smoking is an absolute contraindication for implant placement, but it is still associated with decreased dental implant surveys [14,15].

Bone metabolism is also disturbed directly and indirectly. Smoking directly decreases osteogenesis and angiogenesis and, through this, increases osteoclast cell activity and bone resorption occur. Indirectly, it causes decreased parathormone levels, estrogen production and levels of oxidants. It increases the level of cortisol, as well as free radicals. All of the above have an indirect negative effect on the balance of osteoblasts/osteoclasts activity [16–18], such as bisphosphonates' action [19–21]. Spine densitometry is one of the methods used to check and control the metabolism of human bone [22,23]. Previous studies observed a warning relation between marginal bone loss and the transformation of the bone surrounding dental implants toward corticalization [24,25]. The coexistence of these two phenomena may have an adverse effect on the long-term success of dental implant treatment.

This raised the question of whether a known negative factor (tobacco smoking) increases corticalization.

The aim of this study was to evaluate peri-implant jawbone corticalization in tobacco smokers.

#### **2. Materials and Methods**

This research is the result of analyses of prospective radiological data of oral implantological treatment of patients with dental loss.

The inclusion criteria were as follows: at least 18 years of age, bleeding upon gingival probing < 20%, probing depth ≤ 3 mm, good oral hygiene, regular follow-ups, two-dimensional radiographs taken during regular checks, laboratory tests to check vitamin levels and ions and hormones levels—parathormone (PTH, where the norm is 10 to 60 pg/mL); thyrotropin (TSH), where the norm is 0.23–4.0 μU/mL; calcium in serum (Ca2+), where the norm is 9–11 mg/dL; glycated hemoglobin (HbA1c), where the norm is <5%; and vitamin 25(OH)D3 (D3), where the norm is 31–50 ng/mL. Patients smoking 1 or more cigarettes per day were qualified to be in the smoker group.

Spine densitometry, where the T-score can be examined, was also considered. The T-score shows the ratio between bone mineral density (BMD) of the examined patient and the average BMD for young patients. The normal value for normal bone is >−1.0, osteopenia is indicated by values between −1.0 and −2.5, and scores <−2.5 indicate osteoporosis (Table 1).

**Table 1.** Inclusion criteria for the research.

Smoking 1 or more cigarettes per day

The exclusion criteria included a lack of or defective X-ray images in the visual assessment, lack of laboratory tests, uncontrolled internal co-morbidity (diabetes mellitus, thyroid dishormonoses, rheumatoid disease and other immunodeficiencies), a history of oral radiation therapy, past or current use of cytostatic drugs, soft and bone tissue augmentation, and low-quality or lack of follow-up radiographs. Finally, 2196 samples of a dental implant neck area (768 patients) were included in this study and analyzed (the average number of implants per patient was 2.86) (Table 2).

**Table 2.** Exclusion criteria for the research.

Additionally, the following factors were considered: confidence level of 95%, margin error of 5%, population of 37 million and a fraction of 28.8%. In that case, the minimal sample size is 323 patients. Taking into account the amount of 768 patients, the error is on the level of 3%.

The limitation of the study is that the laboratory tests have not been carried out 3 months after the research (Table 3).

**Table 3.** Limitation of the research.

Twenty-two types of dental implants were used in this study: AB Dental Devices I5 (www.ab-dent.com Ashdod, Israel; accessed on 21 July 2022); ADIN Dental Implants Touareg (www.adin-implants.com Afula, Israel; accessed on 21 July 2022); Alpha Bio ARRP (www.alpha-bio.net Petah-Tikva, Israel; accessed on 21 July 2022); Alpha Bio ATI (www.alpha-bio.net Petah-Tikva, Israel; accessed on 21 July 2022); Alpha Bio DFI (www. alpha-bio.net Petah-Tikva, Israel); Alpha Bio OCI (www.alpha-bio.net Petah-Tikva, Israel; accessed on 21 July 2022); Alpha Bio SFB (www.alpha-bio.net Petah-Tikva, Israel; accessed on 21 July 2022); Alpha Bio SPI (www.alpha-bio.net Petah-Tikva, Israel; accessed on 21 July 2022); Argon K3pro Rapid (www.argon-dental.de Bingen am Rhein, Germany; accessed on 21 July 2022); Bego Semados RI (www.bego-implantology.com Bremen, Germany; accessed on 21 July 2022); Dentium Super Line (www.dentium.com Gyeonggi-do, South Korea; accessed on 21 July 2022); Friadent Ankylos C/X (www.dentsplysirona.com Warszawa, Poland; accessed on 21 July 2022); Implant Direct InterActive (www.implantdirect.com Thousand Oaks, United States of America; accessed on 21 July 2022); Implant Direct Legacy 3 (www.implantdirect.com Thousand Oaks, United States of America; accessed on 21 July 2022); MIS BioCom M4 (www.mis-implants.com Bar-Lev Industrial Park, Israel; accessed on 21 July 2022); MIS C1 (www.mis-implants.com Bar-Lev Industrial Park, Israel; accessed on

21 July 2022); MIS Seven (www.mis-implants.com Bar-Lev Industrial Park, Israel; accessed on 21 July 2022); MIS UNO One Piece (www.mis-implants.com Bar-Lev Industrial Park, Israel; accessed on 21 July 2022); Osstem Implant Company GS III (www.en.osstem.com Seoul, South Korea; accessed on 21 July 2022); SGS Dental P7N (www.sgs-dental.com Schaan, Liechtenstein; accessed on 21 July 2022); TBR Implanté (www.tbr.dental Toulouse, France; accessed on 21 July 2022); and Wolf Dental Conical Screw-Type (www.wolf-dental. com Osnabrück, Germany; accessed on 21 July 2022) (Table 4).

**Table 4.** The implant types used and their features.

Surgery was performed under local anesthesia Artycaine + Adrenaline 1:100,000 by one surgeon (M.K) following the manufacturer-recommended protocols for dental implant placements. The healing process was carried out under a closed mucoperiosteal flap, unloaded in two-stage implants. Implants were loaded after 3 months of healing from the surgery.

Standardized intraoral radiographs were taken immediately after surgery (00M) every 3 months in the first year of healing, every 6 months in the second year and every year until 5 years of observation (5y). Radiographs were taken using the DIGORA OPTIME radiography system (TYPE DXR-50, SOREDEX, Helsinki, Finland). The RTG images were taken in a standardized way [26] with the following parameters: 7 mA, 70 mV, and 0.1 s (the focus apparatus was from Instrumentarium Dental, Tuusula, Finland). Positioners were used to take images repeatably with a 90◦ angle of X-ray beam to the surface of phosphor plate.

Radiologically recorded peri-implant bone structure was studied by digital texture analysis using the Corticalization Index previously proposed [25,27] (as version 1 (CI)). It consists of the product of a measure that evaluates the number of long series of pixels of similar optical density with the mean optical density of the studied site (in the numerator) and the magnitude of the chaotic arrangement of the texture pattern, i.e., differential entropy (in the denominator).

Marginal bone loss (MBL) was measured on radiological images [28] (Figure 1) by only one researcher. Texture of X-ray images was analyzed in MaZda 4.6 freeware invented by University of Technology in Lodz [29,30] to test measures of corticalization in peri-implant environment of trabecular bone (representing original bone before implant-dependent alterations) and soft tissue (representing product of marginal bone loss). MaZda provides both first-order (mean optical density) and second-order (Differential Entropy: DifEntr, Long-Run Emphasis Moment: LngREmph) data. Due to the fact that the second-order data are given for four directions in the image, and in the present study, the authors do not wish to search for directional features, the arithmetic mean of these four primary data was included for further analysis. The regions of interest (ROIs) were marked near the neck area (Figure 2) and normalized (μ ± 3σ) to share the same mean (μ) and standard deviation (σ) of optical density within the ROI. To eliminate noise, [26] further worked on data reduced to 6 bits. For analysis in a co-occurrence matrix, a spacing of 5 pixels was chosen. In the formulas that follow, p(i) is a normalized histogram vector (i.e., histogram whose entries are divided by the total number of pixels in ROI), i = 1, 2,..., Ng, and Ng denotes the number of optical density levels. Mean optical density feature (only a first-order feature) was calculated as below:

$$\text{MeanOptical Density} = \sum\_{i=1}^{N\_{\text{g}}} \text{ip}(i)$$

Second-order features:

$$\text{DiffEntr} = -\sum\_{i=1}^{\text{Ng}} \mathbf{p}\_{\text{x}-\mathbf{y}}(\mathbf{i}) \log(\mathbf{p}\_{\text{x}-\mathbf{y}}(\mathbf{i})) $$

where Σ is the sum; Ng is the number of levels of optical density in the radiograph; i and j are the optical density of pixels with a 5-pixel distance from one another; p is probability; and log is common logarithm [31]. The differential entropy calculated in this way is a measure of the overall scatter of bone structure elements in a radiograph. Its high values are typical for cancellous bone [32–35]. Next, the last primary texture feature was calculated:

$$\text{LngREmph} = \frac{\sum\_{i=1}^{\text{Ng}} \sum\_{k=1}^{\text{Nr}} \mathbf{k}^2 \mathbf{p}(\mathbf{i}, \mathbf{k})}{\sum\_{i=1}^{\text{Ng}} \sum\_{k=1}^{\text{Nr}} \mathbf{p}(\mathbf{i}, \mathbf{k})}$$

where Σ is the sum; Nr is the number of series of pixels with density level i and length k; Ng is the number of levels for image optical density; Nr is the number of pixels in series; and p is probability [36,37]. This texture feature describes thick, uniformly dense, radio-opaque bone structures in intra-oral radiograph images [33,35].

$$\text{CI} = \frac{\text{LngREmph} \cdot \text{Mean Optical Density}}{\text{DiffEntr}}$$

Statistical analysis includes feature distribution evaluation, mean (*t*-test) or median (W-test) comparison, analysis of regression and one-way analysis of variance or Kruskal– Wallis test as non-normal distribution or between-group variance indicated on significant differences in investigated groups. Detected differences or relationships were assumed to be statistically significant when *p* < 0.05. Statgraphics Centurion version 18.1.12 (StatPoint Technologies, Warrenton, VA, USA) was used for statistical analyses.

**Figure 1.** Measuring of marginal bone loss on the radiographic images 5 years after the functional loading. White line indicates the implant platform to the bottom of the bone loss cavity.

**Figure 2.** Marking an ROI. ROIs were marked near the implant neck area. Green area—mesial implant neck area; red area—distal implant neck area; Abbreviations: ROI—region of interest; 00M—0 months of observation; 5y—five years of observation.

#### **3. Results**

#### *3.1. All Samples*

Statistical examination revealed that the initial MBL for the mandible and maxilla in the non-smoker group of samples was lower than in the group with smokers, respectively (mean 0 mm ± 0.85 mm; 0 mm ± 1.13 mm), which was statistically significant (*p* < 0.05). MBL after 5 years of observation was also correlated with nicotinismus according to statistics where *p* < 0.05, meaning there was statistical significance: MBL for non-smokers (mean 0 mm ± 1.25 mm) and MBL for smokers (mean 0.42 mm ± 1.32 mm). Corticalization

Index for smokers and non-smokers was calculated immediately after the implant insertion and 5 years after the implantation; respectively, 185.98 ± 90.8 and 163.97 ± 151.9 for 00M; 243.17 ± 155.47 and 220.32 ± 184.97 after the 5 years. *p* value was lower than 0.05, which means it was statistically significant. Implant loss depending on MBL appearance after five years of observation was checked along with the value and analyzed for smokers and non-smoker group: 1.69 mm ± 1.73 mm and 0 mm ± 1.24 mm. *p* value was lower than 0.05. Implant loss frequency was checked. In a group of smokers, 6.74% of implants were lost; in a group of non-smoking patients, 2.87% of implants were lost, where the *p* value was lower than 0.05 (Table 5, Figure 3).

**Table 5.** Table presents values for marginal bone loss, values of Corticalization Index and implant loss frequency. Values were calculated for all the implantations for smokers and non-smokers. 00M—the observation period immediately after the implantation; 5y—the observation period 5 years after the implantation.

**Figure 3.** Dependences for all samples: (**a**) dependence of marginal bone loss from tobacco smoking after the dental implant insertion after 5 years of observation. The higher marginal bone loss was observed in the tobacco smoker group; (**b**) dependence of Corticalization Index from tobacco smoking after 5 years from implant insertion—there was no statistical difference; (**c**) dependence of implant loss from marginal bone loss after 5 years from functional loading—the higher marginal bone loss, the higher probability of implant loss; (**d**) frequency of implant loss depending on smoker or non-smoker group—the higher frequency was in the smoker group.

#### *3.2. Mandible Group*

Research revealed that the initial MBL for implants in the mandible in the non-smoker group was lower than in the group with smokers, respectively (mean 0 mm ± 0.88 mm; 0 mm ± 1.83 mm), which was statistically significant (*p* < 0.05). MBL after 5 years of observation was also correlated with nicotinismus according to statistics where *p* < 0.05, which means that there was statistical significance: MBL for non-smokers (mean 0 mm ± 1.09 mm) and MBL for smokers (mean 1.10 mm ± 1.46 mm). Corticalization Index for smokers and non-smokers in the mandible implants group was calculated after the implant insertion and 5 years after the implantation, respectively, 195.81 ± 68.8 and 193.27 ± 136.54 for 00M; 263.87 ± 130.7 and 298.02 ± 200.1 after the 5 years. *p* value was higher than 0.05, which means it was not statistically significant. Implant loss depended on MBL appearance after five years of observation in a group of mandible-inserted implants; the value was checked and analyzed for smokers and non-smoker group: 2.36 mm ± 0.94 mm and 0 mm ± 1.13 mm. *p* value was lower than 0.05. The relation between implant loss and the occurrence of corticalization was analyzed. Respectively, for smokers and non-smokers, values for initial corticalization were 183.62 ± 18 and 194.10 ± 131.65; 5 years after the implantation, values were 438.18 ± 341.70 for smokers and 292.40 ± 193.95 for non-smokers, and the *p* value was higher than 0.05. Implant loss frequency was also checked for mandible implants in relation to smoking. In a group of smokers, 1.66% of implants were lost; in a group of non-smoking patients, 1.25% of implants were lost; the relationship was weak, but the *p* value was still lower than 0.05 (Table 6, Figure 4).

**Table 6.** Table presents values for marginal bone loss, values of Corticalization Index and implant loss frequency. Values were calculated for implants in mandible for smokers and non-smokers. 00M—the observation period immediately after the implantation; 5y—the observation period 5 years after the implantation.

#### *3.3. Maxilla Group*

Implants in the maxilla were also examined separately. Statistics showed that the initial MBL for implants in the maxilla in the non-smoker group was lower than in the group with smokers, respectively (mean 0 mm ± 0.82 mm; 0 mm ± 2.38 mm), which was statistically significant (*p* < 0.05). MBL after 5 years of observation was also correlated with nicotinismus according to statistics where *p* < 0.05, meaning there was statistical significance: MBL for non-smokers (mean 0 mm ± 1.29 mm) and MBL for smokers (mean 1.42 mm ± 1.34 mm). The Corticalization Index for smokers and non-smokers in the maxilla implants group was calculated after implant insertion and 5 years after implantation; respectively, 173.50 ± 92.80 and 146.56 ± 109.65 for 00M, where the *p* value was lower than 0.05; 207.06 ± 153.50 and 193.68 ± 151.10 after 5 years. The *p* value was higher than 0.05, meaning there was no statistical significance. Implant loss, depending on MBL appearance after five years of observation in the group of maxilla inserted implants, was checked and analyzed for smokers and non-smoker groups: 1.42 mm ± 1.34 mm and 0 mm ± 1.29 mm. *p* value was lower than 0.05. In this group, the relation between implant loss and the occurrence of corticalization was also analyzed. Respectively, for smokers and non-smokers, values for initial corticalization were 173.56 ± 70.82 and 147.20 ± 109.40; and 5 years after implantation, 235.15 ± 268.71 for smokers and 193.67 ± 147.98 for non-smokers. The

*p* value was higher than 0.05. Implant loss frequency was also checked for maxilla implants in relation to smoking. In the group of smokers, 4.34% of implants were lost; conversely, in the group of non-smoking patients, 1.49% of implants were lost. The relationship between smoking and implant loss was weak, but the *p* value was still lower than 0.05 (Table 7, Figure 5).

**Figure 4.** Dependencies for mandible samples: (**a**) dependence of marginal bone loss on tobacco smoking after the dental implant insertion after 5 years of observation. Higher marginal bone loss was observed in the tobacco smoker group; (**b**) dependence of the Corticalization Index on tobacco smoking 5 years after implant insertion—there was no statistical difference; (**c**) dependence of implant loss on marginal bone loss 5 years after functional loading—the higher the marginal bone loss, the higher probability of implant loss; (**d**) frequency of implant loss depending on the smoker or non-smoker group—the higher frequency was in the smoker group.

**Table 7.** This table presents values for marginal bone loss, Corticalization Index and implant loss frequency. Values were calculated for implants in maxilla for smokers and non-smokers. 00M—the observation period immediately after implantation; 5y—the observation period 5 years after implantation.

**Figure 5.** Dependencies for maxilla samples: (**a**) dependence of the Corticalization Index on tobacco smoking 5 years after implant insertion—there was no statistical difference; (**b**) frequency of implant loss depending on the smoker or non-smoker group—the higher frequency was in the smoker group; (**c**) dependence of implant loss on marginal bone loss a 5 years after functional loading—the higher the marginal bone loss, the higher the probability of implant loss.

#### *3.4. Corticalization vs. Torque*

The study also revealed a correlation between the Corticalization Index and the torque used during implant insertion (when the torque value was increasing), and the probability of occurrence of the corticalization phenomenon was also higher. The *p* value was lower than 0.05 (Figure 6).

**Figure 6.** Dependence of the Corticalization Index on torque value after dental implant insertion, following 5 years of observation. The higher the torque during dental implantation, the higher the Corticalization Index after 5 years.

#### **4. Discussion**

The question that arises is the following: Is corticalization in radiographs associated with a higher risk of bone loss around dental implants in smoking patients? Until now, there have been no publications that can confirm or deny this. In this research, 768 patients with dental implants were studied, and 2196 samples of the neck implant area were analyzed. The study showed that the Corticalization Index is real and changes throughout the healing period, and the value of this index varies depending on the groups analyzed (smoking and non-smoking patients). The presented research shows that there is a close correlation between smoking and changes in bone structure. This correlation was calculated and discovered at the pixel level, taking into account texture features. Marginal bone loss has been analyzed many times, but no one has ever checked the bone structure and its relations to nicotinism at the pixel level [38–40].

Taking into account the corticalization phenomenon in all analyzed implants, a higher index was observed in the group of smokers already at the time of implantation (Table 5). A higher index of corticalization was observed in the group of smokers on the day of surgery, and a smaller index was observed in the group where patients did not smoke (bone changes at the pixel level were observed before implant placement, this may confirm that the tobacco smoking habit leads to undesirable changes in bone morphology). The Corticalization Index increases throughout the healing process. It was also observed in the mandible and maxilla separately, but in the case of the maxilla, the difference was greater. Corticalization is a process that changes the structure of the bone, where the trabecular bone is replaced by cortical bone. Remodeling depends on resorption and new bone formation [41–43]. Cancellous bone is composed of trabeculae with marrow between them. Corticalization is the process that changes trabecular bone into denser tissue—cortical [44].

Marginal bone loss is also correlated with nicotinismus, a fact that is commonly known and confirmed in this research. This study affirmed that marginal bone loss around dental implants is associated with tobacco smoking, as analyzed through radiographs and texture features in a large group of implants. Further research showed that MBL is higher in implants that were inserted in the maxilla in the smoker group, as opposed to implants inserted in the mandible. It can be concluded that smoking has an influence on bone structure before implantation and leads to a higher value of the Corticalization Index. The effect of this correlation may result in marginal bone loss. After five years of observation, the research showed that the higher the MBL, the greater the probability of implant loss in the smoking patients' group, which is indirectly correlated with the occurrence of the corticalization phenomenon [24,25,27].

Directly in statistics, there is no statistical significance between smoking and corticalization, but a similarity can be suspected. The Corticalization Index is consistent after 5 years. Smoking is a factor that can change the structure of bone tissue at the cellular level. Changes depend on disturbances in angiogenesis, impaired vascularization and nutrition of bone cells, with an impact on bone metabolism and osteogenesis. If the tissue is not nourished, it atrophies [45–48]. The study presented shows a close relationship between smoking and MBL and also between smoking and implant failure after 5 years of observation. The research emphasizes the impact of smoking on dental implant loss, mainly in the maxilla. Implant loss frequency was almost two times higher than in the case of mandible implants. All these correlations were detected at the pixel level using only texture features and by calculating and analyzing the Corticalization Index.

Additionally, the appearance of corticalization after 5 years of observation was also correlated with the increasing torque value during the implantation procedure. Recent studies have proved that a high value of torque during implant insertion leads to MBL [49–51]. There is a probability that condensed bone around the dental implant, caused by a high torque value, is not a desirable effect. Currently, the popular preparation method and implantation technique is osseodensification [52–54]. Osseodensification around dental implants increases the primary stability of the implant, but if corticalization may lead to peri-implantitis, marginal bone loss, and consequently dental implant loss, what will be the long-term consequences of densifying bone tissue next to the implant body? This phenomenon should be a focus of future research.

Taking into account the types of implants, it can be stated that not only the design of the dental implant affects the corticalization phenomenon. There is a relationship between bone condition, prosthetic restoration and also the surgeon's experience and preferences [24,55,56].

The corticalization phenomenon, as analyzed in this research, is closely related to smoking, marginal bone loss and implant loss. This phenomenon may be indicated before implant placement and also before marginal bone loss during the control periods when RTG images are taken. Considering the clinical relevance of this research, if there are enough studies about this phenomenon and it becomes well-known, it may become a useful tool for prognosis, contraindications and marginal bone loss prevention. Until now, there have been many tools, as mentioned above, but never at the pixel level [57–59].

Two-dimensional RTG images were taken in a defined period of observation. If the general condition of the patient changes, it may have an impact on bone structure. This can also be observed in the textures of RTG images. A limitation of this study was that laboratory tests were not checked after the implant insertion throughout the observation period. This should be taken into consideration in future research [60].

#### **5. Conclusions**

The higher Corticalization Index that occurs is related to tobacco smoking, even as early as the day of surgery. This is the latest study that confirms the close and non-beneficial impact of the corticalization phenomenon on the implant region, leading to peri-implantitis. This research demonstrated and proved that smoking tobacco has an impact on bone structure, which can be identified at the pixel level without clinical examination, using only two-dimensional radiographs.

The corticalization phenomenon may not be directly related to implant loss after 5 years of observation, but a relationship is observed between corticalization and marginal bone loss after 5 years, which may lead to peri-implantitis and implant loss.

The uniqueness of this research is that all these dependencies can be diagnosed by analyzing texture features in RTG images.

**Author Contributions:** Conceptualization, T.W. and G.T.; data curation, P.H. and T.W.; formal analysis, T.W.; funding acquisition, M.K.; investigation, P.H. and T.W.; methodology, T.W.; resources, T.W.; software T.W.; supervision, M.K.; validation, M.K.; visualization, A.M. and T.W.; writing—original draft, T.W.; writing—review and editing, T.W., M.K. and G.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Medical University of Lodz (grant numbers 503/5-061- 02/503-51-001-18, 503/5-061-02/503-51-001-17, and 503/5-061-02/503-51-002-18).

**Institutional Review Board Statement:** This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of the Medical University of Lodz, PL (protocol no. RNN 485/11/KB and date of approval: 14 June 2011).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data on which this study is based will be made available upon request at https://www.researchgate.net/profile/Tomasz-Wach.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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*Article* **Computer Vision for Detection of Body Posture and Behavior of Red Foxes**

**Anne K. Schütz 1,\* , E. Tobias Krause 2 , Mareike Fischer 3 , Thomas Müller 4 , Conrad M. Freuling 5 , Franz J. Conraths 1 , Timo Homeier-Bachmann 1,† and Hartmut H. K. Lentz 1,†**

**Simple Summary:** Monitoring animal behavior provides an indicator of their health and welfare. For this purpose, video surveillance is an important method to get an unbiased insight into behavior, as animals often show different behavior in the presence of humans. However, manual analysis of video data is costly and time-consuming. For this reason, we present a method for automated analysis using computer vision—a method for teaching the computer to see like a human. In this study, we use computer vision to detect red foxes and their body posture (lying, sitting, or standing). With this data we are able to monitor the animals, determine their activity, and identify their behavior.

**Abstract:** The behavior of animals is related to their health and welfare status. The latter plays a particular role in animal experiments, where continuous monitoring is essential for animal welfare. In this study, we focus on red foxes in an experimental setting and study their behavior. Although animal behavior is a complex concept, it can be described as a combination of body posture and activity. To measure body posture and activity, video monitoring can be used as a non-invasive and cost-efficient tool. While it is possible to analyze the video data resulting from the experiment manually, this method is time consuming and costly. We therefore use computer vision to detect and track the animals over several days. The detector is based on a neural network architecture. It is trained to detect red foxes and their body postures, i.e., 'lying', 'sitting', and 'standing'. The trained algorithm has a mean average precision of 99.91%. The combination of activity and posture results in nearly continuous monitoring of animal behavior. Furthermore, the detector is suitable for real-time evaluation. In conclusion, evaluating the behavior of foxes in an experimental setting using computer vision is a powerful tool for cost-efficient real-time monitoring.

**Keywords:** YOLOv4; computer vision; animal monitoring; animal behavior; animal activity; animal welfare; body posture


*3.2. Fuel*

The raw material processed in the generator were coniferous cuttings—maximum length 10 to 15 cm, maximum width 5 cm, and a maximum thickness of 2 cm. Their typical physiochemical properties are shown in Table 4. The proportion of fine particles and particles with bark in the gasified material was negligible. This fuel was used long-term in this unit. It was available, affordable, and had good quality and properties for gasification. This type of material had a medium agglomeration-slagging propensity [22].

**Table 4.** Properties of used fuel.

r values of original wood sample; d dry sample without moisture.

#### *3.3. Analytical Methods*

Parameters of the fuel from Table 4 (moisture, volatile, and fixed carbon and ash) were measured by thermogravimetric analyzer Netzsch STA 449 F1 Jupiter. The heating rate was 10 ◦C/min in a nitrogen atmosphere up to 900 ◦C. For the amount of ash, there was a change in nitrogen by oxygen and heating continued up to 1200 ◦C. Ultimative analysis of fuel (amount of C, H, O, N and S) was measured by analyzer LECO CHN 628 with added module 628 S. Combustion heat was measured by the calorimeter LECO AC600.

Thermogravimetric analysis (TG) was measured by the analyzer Netzsch STA 449 F1 Jupiter. The heating rate was 10 ◦C/min in nitrogen atmosphere. The differential thermal analysis DTA was measured simultaneously, which measures the temperature difference between the standard sample (aluminium oxide) and the sample during heating.

Composition of the filtration cake inorganic fraction and incrust was measured with apparatus ARL 9400 XP+ equipped with a Rh lamp with a head Be-oxides window.

The distribution of the trapped particle size was performed by a Fritsch Particle Sizer Analysette 22 apparatus, where the light source is an He-Ne-laser with a wavelength of 633 nm and maximum measuring range of 0.1–1250 μm. To ensure the homogeneity of the measured sample and reliable statistics in the illuminated volume, the suspension was permanently mixed in an external mixing device during measurement using ultrasound, which provided for the separation of the agglomerate.

The measuring of organic substances composition trapped by the filtration cake was necessary to solve these substances in the solvent. Mechanically separated cake from the surface of the outer filter layer was inserted into the Soxhlet extractor and washed for four hours by acetone. The obtained solution was injected into the gas chromatograph Hewlett Packard HP 6890 with a mass detector Hewlett Packard MSD 5973 (GC-MS).


**5. Evaluation**

The evaluation was conducted through a series of interviews with specialists in nutrition and physical activity from three different countries: Spain (one male and two females), Mexico (two males and one female) and Costa Rica (one male and two females). *Sensors* **2018**, *18*, 271 11 of 21 **5. Evaluation**

For each interview, a physical prototype and a storyboard were presented to the participant and a series of questions were asked. In each session, the same interview protocol was used. The protocol consisted of three main parts: participants' information, prototype usefulness and prototype usability/ergonomics. The questions in the protocol are detailed in Appendix D. The evaluation was conducted through a series of interviews with specialists in nutrition and physical activity from three different countries: Spain (one male and two females), Mexico (two males and one female) and Costa Rica (one male and two females). For each interview, a physical prototype and a storyboard were presented to the participant and

The goal with this evaluation was to assess the system's perceived usefulness and usability. Moreover, the thoughts of the specialists were gathered and added to the prototypes. a series of questions were asked. In each session, the same interview protocol was used. The protocol consisted of three main parts: participants' information, prototype usefulness and prototype usability/ergonomics. The questions in the protocol are detailed in Appendix D.

The system was presented using the physical device and storyboards [31]. Using storyboards, we presented the main use cases for the prototypes and their benefits. Participants were presented with two storyboards, one for healthy eating behaviors and the other for sportswear reminders. The goal with this evaluation was to assess the system's perceived usefulness and usability. Moreover, the thoughts of the specialists were gathered and added to the prototypes. The system was presented using the physical device and storyboards [31]. Using storyboards,

The developed interview protocol and questionnaire was designed using two main references: The first set of questions (focused on the prototype) were based on different scales [32] with the intention of gather the perspective of the participants on the usefulness of the system. The second part was based on the standard ISO 9241-210:2010 Ergonomics of human-system interaction [33]. we presented the main use cases for the prototypes and their benefits. Participants were presented with two storyboards, one for healthy eating behaviors and the other for sportswear reminders. The developed interview protocol and questionnaire was designed using two main references: The first set of questions (focused on the prototype) were based on different scales [32] with the

To provide a better understanding we will describe our interpretation of the ISO 9241-210:2010 terminology. By effectiveness we mean the accuracy and completeness with which users achieve specified goals. By efficiency we mean resources expended in relation to the accuracy and completeness with which users achieve goals. By satisfaction we mean freedom from discomfort and positive attitudes towards the use of the product. intention of gather the perspective of the participants on the usefulness of the system. The second part was based on the standard ISO 9241-210:2010 Ergonomics of human-system interaction [33]. To provide a better understanding we will describe our interpretation of the ISO 9241-210:2010 terminology. By effectiveness we mean the accuracy and completeness with which users achieve specified goals. By efficiency we mean resources expended in relation to the accuracy and completeness with which users achieve goals. By satisfaction we mean freedom from discomfort and

Conciseness, consistency and self-descriptiveness deal with the amount of information required to deliver the message and the way in which this data is displayed every time. The characteristics of presented information (i.e., detectability, legibility, discriminability, clarity and comprehensibility) are related to freedom from distraction and interpretability. positive attitudes towards the use of the product. Conciseness, consistency and self-descriptiveness deal with the amount of information required to deliver the message and the way in which this data is displayed every time. The characteristics of presented information (i.e., detectability, legibility, discriminability, clarity and comprehensibility) are related to freedom from distraction and interpretability.

Finally, suitable for individualization means that, users can modify interaction and presentation of information to suit their individual needs and suitable for learning means that the system conforms to user's expectations and it prevents or tolerates errors. Finally, suitable for individualization means that, users can modify interaction and presentation of information to suit their individual needs and suitable for learning means that the system conforms to user's expectations and it prevents or tolerates errors.

#### **6. Results and Discussion 6. Results and Discussion**

In this section, we present the results of the interviews. The evaluation protocol was explained in the Evaluation Section. All results are positive. However, some of the evaluators provided feedback and improvement opportunities that we will also discuss. We asked the evaluators to use three words to describe the system. Figure 4 shows the results of this exercise. The most common word used to describe the system was useful (5 out of 9) followed by simple (4 out of 9). In this section, we present the results of the interviews. The evaluation protocol was explained in the Evaluation Section. All results are positive. However, some of the evaluators provided feedback and improvement opportunities that we will also discuss. We asked the evaluators to use three words to describe the system. Figure 4 shows the results of this exercise. The most common word used to describe the system was useful (5 out of 9) followed by simple (4 out of 9).

**Figure 4. Figure 4.** Word cloud of main characterizers used by evaluators. Word cloud of main characterizers used by evaluators.

Some words caught the interviewer's attention. For instance, when a reviewer used the word "accessible", we followed up to determine that she was referring to the possibility of many people to reach the information on the device. This includes everyone in the household and since the system uses graphical representations it is also accessible for children that do not know how to read or write. *Sensors* **2018**, *18*, 271 12 of 21

One of the interviewees also used the word "basic" we followed up to determine that she saw potential to add other functionalities to the device. Therefore, the version presented was too basic. Another word was "organized" delving in this we determined that the characterization was for the users and not the system. Therefore, organized people are potential users of the system. Some words caught the interviewer's attention. For instance, when a reviewer used the word "accessible", we followed up to determine that she was referring to the possibility of many people to reach the information on the device. This includes everyone in the household and since the system uses graphical representations it is also accessible for children that do not know how to read or write.

Two questions were asked to the evaluators to assess their willingness to either use the system or to recommend it to their friends, family or patients. Figure 5 shows the answers to this question. It is interesting to observe that nine out of nine evaluators (specialists in nutrition and physical activity) are somewhat interested in using the system. However, eight out of nine evaluators consider that they would recommend the system. To further explain this, we must note that usually specialists in nutrition and physical activity have a healthy lifestyle, therefore they might not see fit to use the system. However, during the interviews most of the evaluators stated that remembering healthy snacks was one of the main problems of their patients. One of the interviewees also used the word "basic" we followed up to determine that she saw potential to add other functionalities to the device. Therefore, the version presented was too basic. Another word was "organized" delving in this we determined that the characterization was for the users and not the system. Therefore, organized people are potential users of the system. Two questions were asked to the evaluators to assess their willingness to either use the system or to recommend it to their friends, family or patients. Figure 5 shows the answers to this question. It is interesting to observe that nine out of nine evaluators (specialists in nutrition and physical activity) are somewhat interested in using the system. However, eight out of nine evaluators consider that they would recommend the system. To further explain this, we must note that usually specialists

Some of the evaluators stated that they would not use the system as it is. But they would reconsider if it included some of their feedback (especially screen size and the amount of information that can be delivered through the device). The evaluation had two main focuses, the first one was to assess the evaluator's opinion on the system, the second one was focused on the look and feel of the device (human factors and ergonomics perspective). We asked a question regarding the evaluators' satisfaction with the look and feel of the devices. All the answers were positive (5 somewhat satisfied, 3 very satisfied and 1 extremely satisfied). Figure 6 shows the responses to this question. in nutrition and physical activity have a healthy lifestyle, therefore they might not see fit to use the system. However, during the interviews most of the evaluators stated that remembering healthy snacks was one of the main problems of their patients. Some of the evaluators stated that they would not use the system as it is. But they would reconsider if it included some of their feedback (especially screen size and the amount of information that can be delivered through the device). The evaluation had two main focuses, the first one was to assess the evaluator's opinion on the system, the second one was focused on the look and feel of the device (human factors and ergonomics perspective). We asked a question regarding the evaluators'

Most of the improvement opportunities mentioned by the evaluators include: enlarging the device screen and making it in color, changing the shape of the case, adding sound to the notifications, and adding text to the notification and not only images. This were the main concerns regarding the device. satisfaction with the look and feel of the devices. All the answers were positive (5 somewhat satisfied, 3 very satisfied and 1 extremely satisfied). Figure 6 shows the responses to this question. Most of the improvement opportunities mentioned by the evaluators include: enlarging the device screen and making it in color, changing the shape of the case, adding sound to the notifications, and adding text to the notification and not only images. This were the main concerns regarding the

One of the evaluators recommended to add text, this text would be used to provide information about the ingredients of the snack. In Costa Rica, the two of the evaluators asked to change the shape of the snack reminder case to an apple as it is traditional in Costa Rica to have an apple shaped magnet fridge. device. One of the evaluators recommended to add text, this text would be used to provide information about the ingredients of the snack. In Costa Rica, the two of the evaluators asked to change the shape of the snack reminder case to an apple as it is traditional in Costa Rica to have an apple shaped magnet fridge.

**Figure 5. Figure 5.** Likelihood to use the system or recommend the system. Likelihood to use the system or recommend the system.

Another important observation was that some of the evaluators compared the system with mobile applications with similar functionalities. Other evaluator requested for the system to sync with mobile applications. Another important observation was that some of the evaluators compared the system with mobile applications with similar functionalities. Other evaluator requested for the system to sync with mobile applications.

*Sensors* **2018**, *18*, 271 13 of 21

Delving on the device functionalities, most of the comments were focused on the snacking reminder device. The first recommendation was to add reminders not only to people to carry their snacks but also to prepare them in advance. This would be helpful to assure that the snacks are ready to go when they are required. One evaluator recommended to use only one device with more information (i.e., combining both devices). Delving on the device functionalities, most of the comments were focused on the snacking reminder device. The first recommendation was to add reminders not only to people to carry their snacks but also to prepare them in advance. This would be helpful to assure that the snacks are ready to go when they are required. One evaluator recommended to use only one device with more information (i.e., combining both devices).

The last part of the evaluation focused on the ergonomics of human-system interaction. In this section, 13 characteristics of the system were assessed by the evaluators. Figure 7 shows the results of this evaluation segmented by country. Table 4 shows the five-number summary and a reliability analysis of the data depicted in Figure 7. The last part of the evaluation focused on the ergonomics of human-system interaction. In this section, 13 characteristics of the system were assessed by the evaluators. Figure 7 shows the results of this evaluation segmented by country. Table 4 shows the five-number summary and a reliability analysis of the data depicted in Figure 7.

**Figure 6.** Evaluator's satisfaction with the look and feel of the system. Scale (0, 3, 6, 0) represents the number of participants in each category. **Figure 6.** Evaluator's satisfaction with the look and feel of the system. Scale (0, 3, 6, 0) represents the number of participants in each category.

An interesting finding of this evaluation is that both Mexico and Costa Rica on average consider the system good (4.3 out of 5 points). However, in Spain, the opinions were less positive (3.9 out of 5 points). We do believe that being Latin-American countries, Costa Rica and Mexico share opinions on how the system could be used. An interesting finding of this evaluation is that both Mexico and Costa Rica on average consider the system good (4.3 out of 5 points). However, in Spain, the opinions were less positive (3.9 out of 5 points). We do believe that being Latin-American countries, Costa Rica and Mexico share opinions on how the system could be used.

In general, the best ranked categories were system's comprehensibility, the perceived effectiveness and clarity (4.5 out of 5 points). The worst ranked categories are the system's suitability for learning and it discriminability (3.7 out of 5 points). In general, the best ranked categories were system's comprehensibility, the perceived effectiveness and clarity (4.5 out of 5 points). The worst ranked categories are the system's suitability for learning and it discriminability (3.7 out of 5 points).

with mobile applications.

information (i.e., combining both devices).

analysis of the data depicted in Figure 7.

number of participants in each category.

on how the system could be used.

Another important observation was that some of the evaluators compared the system with mobile applications with similar functionalities. Other evaluator requested for the system to sync

Delving on the device functionalities, most of the comments were focused on the snacking reminder device. The first recommendation was to add reminders not only to people to carry their snacks but also to prepare them in advance. This would be helpful to assure that the snacks are ready to go when they are required. One evaluator recommended to use only one device with more

The last part of the evaluation focused on the ergonomics of human-system interaction. In this section, 13 characteristics of the system were assessed by the evaluators. Figure 7 shows the results of this evaluation segmented by country. Table 4 shows the five-number summary and a reliability

**Figure 6.** Evaluator's satisfaction with the look and feel of the system. Scale (0, 3, 6, 0) represents the

An interesting finding of this evaluation is that both Mexico and Costa Rica on average consider the system good (4.3 out of 5 points). However, in Spain, the opinions were less positive (3.9 out of 5 points). We do believe that being Latin-American countries, Costa Rica and Mexico share opinions

In general, the best ranked categories were system's comprehensibility, the perceived

**Figure 7. Figure 7.** Evaluation of system characteristics based on the standard ISO 9241-210:2010. Evaluation of system characteristics based on the standard ISO 9241-210:2010.

**Table 4.** Minimum, maximum, quartiles and reliability analysis for Likert scales depicted in Figure 7. All items Cronbach Alpha: 0.8307, Std. Alpha: 0.8431. Reliability analysis calculated using [34].

A system is suitable for learning when it supports and guides the user in learning to use the system. In our case since the notification and device are supposed to share little information the suitability for learning suffers. To be able to learn to use the system a simple guide should be provided. The discriminability problem is mostly related to the device screen size and resolution. The implemented prototype used a 0.96-inch monochrome OLED graphic display; to improve this, a bigger screen should be used. Moreover, images that avoid ambiguity could be used. Enlarging the screen would also allow for text to be shown, improving discriminability of the notifications.

The final improvement opportunities for the system are focused on the notifications. One evaluator suggested adding personalization based on the user's data and preferences (already considered in the system). However, she added the possibility to add diseases and allergies into the notifications. Another evaluator suggested the necessity of creating profiles (also considered in the system), but he requested to be able to change between profiles using the device.

In general, one recommendation is to add more nutrition facts of the snacks. For instance, alimentary groups, portion sizes and water consumption.

One confronted opinion between evaluators was if the personalization should be performed by the user or if the definition of snacks should be personalized by the nutritionist. One of the evaluators suggested that she would like to be able to specifically set the food characteristics for a given day in their patient's device. Another evaluator suggested doing a consumption analysis of the user. However, this would require sensing capabilities that the system does not provide as it is designed for notifications.

The last opinion on the system is that two evaluators would prefer this to be a mobile application rather than a physical device. The main reason provided is that most people already have a mobile device. During the design phase of this system, this discussion was carried.

One of the main arguments to avoid push notifications on smart devices is that the indiscriminate use of these notifications is causing people to stop paying attention to them. Social media notifications, dedicated apps (e.g., Netflix, Yelp, and Amazon), email, and other mobile phone notifications are just some examples of this overwhelming amount of notifications.

Finally, all evaluators highlighted the positive effect of using these devices in day to day to prevent overweight and obesity if activities and tasks associated with notifications are carried out properly.


**Table 1.**

*The chemical and physical properties of cement and silica fume [18].*

**Figure 1.** *Particle size distributions of silica fume [18].*

taken from the region of Bou Sâada (250 km east of Algiers). The sieve analysis is performed according to the European standard NF EN 933-1. The mineralogical composition determined by X-ray diffraction shows that the siliceous sand is more than 95% of quartz and calcite traces.

The coarse fraction of aggregate is gravel (G1) of size 3/8 mm and gravel (G2) of size 8/15 mm. The adjuvant used is a super plasticizer which is highly water reducing (Medaplast SP40). It is a solution of pH = 8.2 and a density of 1.22, with 40% of solids. Its normal use scale is fixed by the manufacturer's recommendation which is between 0.6 and 2.5% of the cement weight. The tap water used all through the study from mixing was taken from the laboratory of civil engineering.

### **3.2 Mixture design**

Fresh concrete mixes were prepared in a modified laboratory mixer; the mixing procedure is explained in **Table 2**. The concrete specimens were preserved in their molds in a wet place at a temperature of 20°C and 95% relative humidity (RH) during 24 h. After demolding, they were immersed in water at 20°C until the age of testing. The physical and mechanical characteristics of the concretes with and without the addition of silica fume have been compared. The silica fume is added at a dosage of 5% cement weight and the super plasticizer at 2 and 2.5%, respectively. The final compositions of high-performance concrete (HPC), after optimization, are reported in **Table 3** [19, 22].

The following acronyms will be used henceforth:

CR2.5: Concrete without silica fume and 2.5% (by weight of cement) of the chemical admixture.

HPC2.5: Concrete dosed at 5% of silica fume and 2.5% of the chemical admixture. HPC2: Concrete dosed at 5% of silica fume and 2% of the chemical admixture.

**113**

*Mechanical Behavior of High-Performance Concrete under Thermal Effect*

After 28 days, specimens with dimensions (100 mm × 100 mm × 100 mm3

**) Cement Sand Gravel (3/8, 8/15) Water Additions**

CR2.5<PHONE_NUMBER> 220 — 2.5 HPC2.5<PHONE_NUMBER> 119 5 2.5 HPC2<PHONE_NUMBER> 122 5 2

dried in an oven (at 100 °C), until stabilization of their mass. All specimens are subjected to high temperatures, 200, 400, 600, and 900°C, according to the timetemperature schedule of ASTM E 119-00 [39]. After cooling, they were subjected

The slump values were obtained for all three mixtures according to the NF EN 12390-4 and EN 12390-5 [40, 41]. The axial compressive strength was tested at 28 days according to NF EN 12390-4 [40] for the concrete at 20°C that was not

The protocol of porosity accessible to water conforms the recommendations of AFREM group [42]. The open porosity allows us to appreciate the evolution of hydration and structuration of hydrated products; this is a key for identification of the most sustainable concrete [43]. The test pieces for testing of water porosity are dried in an oven at a temperature of 100°C to constant weight and then returned to

The porosity test is carried out on test pieces of dimensions 10 × 10 × 10 cm3

• Dry in an oven at 105°C the sample for at least 24 h until obtaining a constant

• Heat to boil for 5 h, and then weigh the sample in air (i.e., the weight is denoted

C − A

Phase compositions of these concretes were investigated on the fine powders using X-ray diffraction method. The powder samples of concrete heat-treated aggregates at 20, 600, and 900°C were collected after abrasion. X-ray diffraction analysis was performed on an X-ray diffractometer (X'Pert) coupled to a computer system. The essential purpose of this analysis is to identify the different phases of

Gravimetric and differential thermal analyses (ATG and ATD) make it possible to quantify portlandite; these techniques are used to characterize degradations.

C − D× 100 (1)

P (%) = \_

) are

**Silica fume (%) Super plasticizer (%)**

, by

*DOI: http://dx.doi.org/10.5772/intechopen.89916*

*Compositions of concrete with and without silica fume [18].*

to compression tests.

**Mix (kg/m3**

**Table 3.**

subjected to high temperatures.

room temperature in a desiccator.

mass. This mass is denoted as "A."

• Immerse the sample in water for 24 h.

The porosity was calculated by the formula:

• Then hydrostatic weigh (the weight is denoted as "D").

applying the following steps:

as "C").

crystal present in a sample.

**Table 2.**

*Mixing procedure [18].*

#### **Table 3.**

*Compressive Strength of Concrete*

than 95% of quartz and calcite traces.

*Particle size distributions of silica fume [18].*

**3.2 Mixture design**

**Figure 1.**

are reported in **Table 3** [19, 22].

chemical admixture.

The following acronyms will be used henceforth:

60 Mixing of aggregates, silica fume, cement

30 Addition of the remaining super plasticizer

**Time,s Mixing procedure**

180 Mixing

60 Mixing

taken from the region of Bou Sâada (250 km east of Algiers). The sieve analysis is performed according to the European standard NF EN 933-1. The mineralogical composition determined by X-ray diffraction shows that the siliceous sand is more

study from mixing was taken from the laboratory of civil engineering.

The coarse fraction of aggregate is gravel (G1) of size 3/8 mm and gravel (G2) of size 8/15 mm. The adjuvant used is a super plasticizer which is highly water reducing (Medaplast SP40). It is a solution of pH = 8.2 and a density of 1.22, with 40% of solids. Its normal use scale is fixed by the manufacturer's recommendation which is between 0.6 and 2.5% of the cement weight. The tap water used all through the

Fresh concrete mixes were prepared in a modified laboratory mixer; the mixing procedure is explained in **Table 2**. The concrete specimens were preserved in their molds in a wet place at a temperature of 20°C and 95% relative humidity (RH) during 24 h. After demolding, they were immersed in water at 20°C until the age of testing. The physical and mechanical characteristics of the concretes with and without the addition of silica fume have been compared. The silica fume is added at a dosage of 5% cement weight and the super plasticizer at 2 and 2.5%, respectively. The final compositions of high-performance concrete (HPC), after optimization,

CR2.5: Concrete without silica fume and 2.5% (by weight of cement) of the

30 Addition 100% of water and a third of the volume of super plasticizer

HPC2.5: Concrete dosed at 5% of silica fume and 2.5% of the chemical admixture. HPC2: Concrete dosed at 5% of silica fume and 2% of the chemical admixture.

**112**

**Table 2.**

*Mixing procedure [18].*

*Compositions of concrete with and without silica fume [18].*

After 28 days, specimens with dimensions (100 mm × 100 mm × 100 mm3 ) are dried in an oven (at 100 °C), until stabilization of their mass. All specimens are subjected to high temperatures, 200, 400, 600, and 900°C, according to the timetemperature schedule of ASTM E 119-00 [39]. After cooling, they were subjected to compression tests.

The slump values were obtained for all three mixtures according to the NF EN 12390-4 and EN 12390-5 [40, 41]. The axial compressive strength was tested at 28 days according to NF EN 12390-4 [40] for the concrete at 20°C that was not subjected to high temperatures.

The protocol of porosity accessible to water conforms the recommendations of AFREM group [42]. The open porosity allows us to appreciate the evolution of hydration and structuration of hydrated products; this is a key for identification of the most sustainable concrete [43]. The test pieces for testing of water porosity are dried in an oven at a temperature of 100°C to constant weight and then returned to room temperature in a desiccator.

The porosity test is carried out on test pieces of dimensions 10 × 10 × 10 cm3 , by applying the following steps:

The porosity was calculated by the formula:

$$
\begin{aligned}
\text{by the formula:}\\\\
\text{P (\%)} &= \frac{\text{C} - \text{A}}{\text{C} - \text{D}} \times 100\end{aligned}
\tag{1}
$$

Phase compositions of these concretes were investigated on the fine powders using X-ray diffraction method. The powder samples of concrete heat-treated aggregates at 20, 600, and 900°C were collected after abrasion. X-ray diffraction analysis was performed on an X-ray diffractometer (X'Pert) coupled to a computer system. The essential purpose of this analysis is to identify the different phases of crystal present in a sample.

Gravimetric and differential thermal analyses (ATG and ATD) make it possible to quantify portlandite; these techniques are used to characterize degradations.


**4. Conclusions**

Our results confirm that Cu/gCN nanomaterial composites can be useful for the development of SERS/SERRS applications. Our Cu NPs on sp2-bonded carbon nitride supports were fabricated using pulsed laser ablation of a Cu plate immersed in acetonitrile solvent. We carried out chemical, structural, and spectroscopic analyses to study the nature of the Cu NPs and their gCN support. The N:C ratio of the gCN material determined using bulk analysis was ≈1.3, significantly lower than the semiconducting carbon nitride compounds based on heptazine- to triazine-based structures, but it contained close-to-layered gCN materials containing mixed C–N/C–C bonding prepared using vapor deposition techniques. Such materials might contain locally electronically conducting domains. The degree of layer polymerization was incomplete, as shown by strong IR and Raman signals from terminal C≡N bonds. The Cu/gCN hybrid nanocomposites exhibited strong visible absorption extending toward the near-IR region with an SPR signal from the metal NPs at 580 nm. SERS/SERRS enhancement activity was tested using three analyte molecules (CV, MB, and R6G) prepared with initial solution concentrations of 10−3–10−7 mol/L in acetonitrile and adsorbed onto the Cu/gCN nanocomposite surfaces. The observed SERS/SERRS enhancement factors were on the order of 107, comparable with *EF* values found for nanoparticle arrays of Cu produced using sputtering onto templates and other noble metal NPs supported by gCN materials. Further studies are now needed to investigate the possibility of cooperative interactions between the gCN support and the metal NPs that could enhance the optoelectronic effects leading to SERS/SERRS enhancement, as well as in stabilizing the Cu NPs.

**Supplementary Materials:** The Supplementary Materials are available online at http://www.mdpi.com/2079-4991/ 9/9/1223/s1.

**Author Contributions:** The study was devised by R.M. and synthesis, Raman, and SERS experiments were performed by H.D.-A. TEM and XPS characterization was performed by T.S.M. All authors participated in the interpretation of the results. H.D.-A. wrote a first draft of the manuscript that was completed and edited by P.F.M. and T.S.M.

**Funding:** R.M. thanks the Iran Nanotechnology Initiative Council (INIC) for financial support of the pulsed laser ablation setup. P.F.M. acknowledges support from the EU Graphene Flagship under Horizon 2020 Research and Innovation program grant agreement No. 696656—Graphene Core2. T.S.M. thanks the UK Engineering Physical Research Council for support via the EPSRC Postdoctoral Fellowship EP/P023851/1.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.


**2. Materials and Methods**

The experimental work presented in this paper was conducted using a wave flume at the Sir John Laing Building, Coventry University (Figure 1). The flume is 18 m long, 1 m deep, and 0.6 m wide. A wave generator is located at the upstream end of the flume while a beach is located at the downstream end to dissipate the energy induced by the waves reproduced.


**Author details**

Jung-Ran Park\*, Andrew Brenza and Lori Richards The College of Computing and Informatics, Drexel University, Philadelphia, USA

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 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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