*4.1. The E*ff*ect of Strontium Concentration in Solution on Phase Composition of the Precipitate*

The strontium content in the solution significantly affects the phase composition of the precipitate. Considering the absence of Sr2+ ions in the solution of non-stoichiometric composition (Ca/C2O4 = 5) containing citric acid, tetragonal calcium oxalate dihydrate weddellite (Figures 1a and 2a) is obtained, which is unstable in the crystallization field of monoclinic calcium oxalate monohydrate whewellite [17,21]. The addition of a small amount of Sr2+ ions (Sr/(Sr + Ca) = 5%) to this solution violates the conditions favorable for weddellite crystallization and leads to intensive crystallization of whewellite, the content of which in the precipitate exceeds the amount of weddellite by 2.6 times (Figures 1b and 2b, Table 1). Accompanying an increase in the strontium content in the solution, the amount of weddellite in the precipitate gradually increases and, at a ratio of Sr/(Sr + Ca) = 35–45%, matches the amount of whewellite (Figures 1c and 2d). A further increase in strontium content again creates conditions favorable for the crystallization of weddellite, the content of which in the precipitate begins to prevail (Figures 1d and 2e). Reaching an Sr/(Sr + Ca) ≥ 80% ratio in solution, the precipitate consists almost exclusively of weddellite. Thus, the presence of strontium in the crystallization medium can be favorable for the crystallization of both weddellite and whewellite, depending on the concentration.


**5.2 Fermentation**

Microorganisms are employed to metabolize the liberated single sugars from enzymatic hydrolysis to convert them to bioethanol. There are two approaches:

SSF is currently considered the optimal method to convert lignocellulose to bioethanol. The process is reported with high conversion yield [21]. However, there are still some small backwards of this method. The optimal temperature for enzymatic hydrolysis is 45–50°C, while fermentation is at its highest efficiency at 28–35°C. Moreover, some intermediate products also resist the growth of microorganisms [25, 38].

Different microorganisms can be employed to enhance the fermentation. **Table 3** presents popular microorganisms, which can metabolize sugars and excrete ethanol.

From **Table 3**, it is easy to understand why *Saccharomyces cerevisiae* is a favorable choice of yeast to ferment sugar solution to bioethanol. Thanks to its tolerance to high ethanol concentration and material's inhibitors, *Saccharomyces cerevisiae* is known not only a traditional but also the most popular yeast in bioethanol production.

In fermentation process, an additional nutrient is necessarily added to provide organic nitrogen source for the growth of microorganisms. Peptone, corn steep liquor (CSL), urea, and even the distillation residue of bioethanol production process have been employed and investigated [32].

Production of lignocellulosic ethanol is still cost-inefficient. In attempts to improve bioethanol fermentation yield, more than one microorganism strain can

#### **Table 3.**

*Some popular microorganisms for bioethanol production [39].*

be loaded to the fermentation mixture as simultaneous saccharification and cofermentation (SSCF) method [39]. Hereby, both hexose and pentose can be utilized to produce bioethanol.


**5. Summary and conclusions**

Transcatheter aortic valve replacement (TAVR) is a minimally invasive treatment for those patients with severe aortic stenosis who cannot be treated surgically due to surgical risk. The first human experience was demonstrated in 2002 and after this date, it started to be performed all over the world. As the experience on this subject increases, TAVR has been brought to the agenda in patients with intermediate and low surgical risk, and studies on this subject are continuing. The procedure is generally performed by femoral approach. Different approaches (transapical, transaortic, subclavian/axillary, transcarotid, suprasternal and transcaval) can be used in patients who are not anatomically suitable for the femoral approach. Venous access was also obtained with two arterial access. A temporary pacemaker is placed via the venous route. Main arterial site is for delivery system and second site is for pigtail. Two types of valves can be used in the TAVR process: self-expandable (SE) and balloon-expandable (BE). In this chapter, we discuss valve types, which valve type will be preferred in which patient, the technical parts of different accesses and the specific complications of the procedures, and the points to be considered during the procedure.


**Abstract**

Different means of hydrological data collection have developed and used. However, they are constraint in one way or other. This paper therefore revisited the rainfall simulator as potential tool for hydrological research. The research disclosed that there are three different types of rainfall simulators; drop former simulator, pressure nozzle simulator and hybrid simulator. It can further be classified as indoor model and outdoor. The research also showed that precipitation is the driving force in hydrological studies. Consequently, in the design of rainfall simulator, the following should be taken into consideration: nozzle spacing, pump size, nozzle size, nozzle type, nozzle spacing, plot size and pressure. Meanwhile, intensity, distribution uniformity, kinetic energy, rainfall drop size and rainfall terminal velocity should be noted in its evaluation. Factoring-in the aforementioned design considerations, data collection is made easy without necessarily waiting for the natural rainfall. Since the rainfall can be controlled, the erratic and unpredictable changeability of natural rainfall is eliminated. Emanating from the findings, pressurized rainfall simulator produces rainfall characteristics similar to natural rainfall, which is therefore recommended for laboratory use if natural rainfall-like characteristics is the main target.

**Keywords:** rainfall-simulator, intensity, uniformity, kinetic-energy, drop-size, runoff, hydrology, research-tool

### **1. Introduction**

Disintegration of the soil are impelled by the effect of rain drops on plain or almost plain soils, which detaches and splash soil particles and transports them downslope as a feature of surface flow. The net disintegration rate (sediments mass/unit zone) is an element of both rain sprinkle and surface flow. Runoff from earth surface conveys with it the most erodible sediment and fine sand particles from the dirt surface as the water streams downhill. At that point, rills are shaped; they start little channels, inevitably framing gaps, which can bring about enormous soil losses [1]. These processes are regularly studied in the field with normal precipitation which may be moderated by uncontrolled factors such as irregularity of the precipitation events. This paper therefore reviewed rainfall simulator as a potential tool for hydrological research.


*Review* **An Overview on Fault Diagnosis, Prognosis and Resilient Control for Wind Turbine Systems**

**Zhiwei Gao 1,\* and Xiaoxu Liu 2**

**Abstract:** Wind energy is contributing to more and more portions in the world energy market. However, one deterrent to even greater investment in wind energy is the considerable failure rate of turbines. In particular, large wind turbines are expensive, with less tolerance for system performance degradations, unscheduled system shut downs, and even system damages caused by various malfunctions or faults occurring in system components such as rotor blades, hydraulic systems, generator, electronic control units, electric systems, sensors, and so forth. As a result, there is a high demand to improve the operation reliability, availability, and productivity of wind turbine systems. It is thus paramount to detect and identify any kinds of abnormalities as early as possible, predict potential faults and the remaining useful life of the components, and implement resilient control and management for minimizing performance degradation and economic cost, and avoiding dangerous situations. During the last 20 years, interesting and intensive research results were reported on fault diagnosis, prognosis, and resilient control techniques for wind turbine systems. This paper aims to provide a state-of-the-art overview on the existing fault diagnosis, prognosis, and resilient control methods and techniques for wind turbine systems, with particular attention on the results reported during the last decade. Finally, an overlook on the future development of the fault diagnosis, prognosis, and resilient control techniques for wind turbine systems is presented.

**Keywords:** wind turbine; energy conversion systems; condition monitoring; fault diagnosis; fault prognosis; resilient control


**2.2 Fourier transform infrared (FTIR) spectroscopy**

The IRTrace-100 FTIR spectrophotometer (Shimadzu, Kyoto, Japan) was used for the FTIR analysis. The extracted celluloses were analyzed to investigate the difference in the functional groups after extraction. The spectral results were recorded within a range of 500–4000 cm1 wavelength using 4 cm1 spectral resolution and 34 scans. **Figure 1** shows several major absorption bands and the difference between the samples. DPW sample before isolation showed typical lignocellulose strong band absorption bonds. For instance, the bands at 1037 cm1

### **Table 1.**

*Physicochemical analysis of biomass (dried basis).*

MJkg1 more energy than lithium ion batteries (0.8 MJkg1

*Biotechnological Applications of Biomass*

suggested [10, 11].

multiple heating rates [13].

**580**

biomass is considered a potential candidate to sustainable green alternative source of energy and chemicals due to its high energy density, volatile matter content, and global widespread [3]. The release of volatile matter and other contents in biomass has been extensively studied using pyrolysis technology. Pyrolysis involves the conversion of biomass into bio-oil, gases (volatile matter) and biochar, in the absence of oxygen [4, 5]. The technique is robust and essential in providing vital knowledge of kinetics of devolatilization of any biomass prior to further processing via different conversion technologies. In addition, pyrolysis is effective in reducing the bulky biomass into uniform, energy dense, and easily transportable fuel [6]. Despite always being the first stage in most combustion or gasification process, there are no accurate and enough data on the kinetics and reaction mechanisms of different lignocellulosic biomass [7]. This is attributed to the complexity and the varying physico-chemical properties in different lignocellulosic biomass [7]. In addition, there may be many reactions occurring from the extremely complex pyrolysis process of the lignocellulose biomass [8]. Therefore, developing accurate kinetic models to account for all reactions taking place remains a challenge [6]. Isolation of the complex lignocellulosic biomass into individual fractions and characterization of the individual fractions can provide a better understanding of the combustion kinetics and reaction mechanism. Different biomass isolation/extraction techniques such as liquid-liquid, liquid-solid, acid-base, ultrasound, and microwave-assisted extractions, among others, have been reported before [9]. The choice of the method depends on the biomass type and its fraction to be isolated [9]. Hence, each procedure affects the sample's product yields and physical, chemical, kinetic, and thermodynamic properties differently. Despite the studies on the yields and operating parameters such as solvent and time [9], less or no information is available regarding the kinetic and thermodynamic parametric studies for the combustion of the isolated lignocellulose fractions to assess the difference in the extraction processes. Different general kinetic models on lignocellulose biomass have been

Date palm waste constitutes about 500,000 metric tons per year from ca. 44 million date palm trees found in the United Arab Emirates where this research was conducted. The aims of this research are to isolate cellulose from date palm lignocellulose complex using low concentration acid-base solutions and Organosolv techniques and to model nonisothermal combustion kinetics using model-free methods and finally to predict the most probable mechanistic reaction mechanism of the isolated celluloses. Using thermal-gravimetric technique at different heating rates, kinetic and thermodynamic parameters were calculated using model-free methods, namely Kissinger-Akahira-Sunose (KAS), Flynn-Wall-Ozawa (FWO), and Starink models. The FWO model-free method compensates the experimental measurement errors. However, the KAS and Starink methods depend on the choice of good constant degree of conversion from the derivative mass loss function to provide precision of the kinetic data [12]. In addition, application of different model-free methods involves wide conversion range that allows for study of change in mechanism during a reaction and reduces mass transfer limitations by using

**2. Isolation techniques and nonisothermal kinetic studies**

The rachis part of adult date palm waste (DPW) (10–15 years old) was supplied by the UAE University farm, Al foah, Al Ain. The samples were ground to 180 micron particle size to reduce the effects of heat and mass transfer limitations. All

) [3]. Lignocellulose

**Figure 1.** *FTIR spectrum of (a) lignocellulose DPW, (b) Organosolv cellulose and (c) acid-base cellulose.*

indicating CdO, C]C and CdC bond stretching, between 2840 and 2926 cm1 indicating CdH stretching and 3200–3474 cm1 for OdH stretching, were assigned to cellulose, hemicellulose and lignin, respectively. Similar results were reported for other biomass in the literature [15]. However, there were noticeable changes in the functional groups of celluloses from the same biomass with reduction in the peak intensity, an indication of component(s) removal (plausibly lignin and hemicellulose). For instance, the would-be lignin and hemicellulose band intensity at 1037 cm1 greatly reduced an indication of component(s) removal. The CdH bond stretching in the region of 2840 assigned to lignin in DPW was absent in isolated cellulose samples. In addition, the reduced peak intensity between 845 and 1156 cm1 associated with the CdOdC asymmetrical stretching and glycosidic bond, a characteristic of cellulose, was observed. Furthermore, the decrease in OH vibration strength around 3200–3474 cm1 indicates a reduction in some of the OHcontaining compounds which are phenolics from lignin. It is worth to note that both extraction methods showed similar functional groups except that the Organosolv cellulose had CdH bond assigned to lignin in the region around 2326 and 2363 cm1 which was absent for the acid-base cellulose samples. The FTIR results showed the effectiveness of the cellulose isolation methods from DPW biomass complex. The samples were further characterized for their morphological differences using SEM imaging technique.

and hemicellulose. Acid-base cellulose in **Figure 2(b)** showed porous surface similar to those reported for cellulose from the teak wood [16]. However,

*SEM images of the (a) original rachis, (b) acid-base cellulose and (c). Organosolv cellulose captured at*

*Investigation of Nonisothermal Combustion Kinetics of Isolated Lignocellulosic Biomass…*

*DOI: http://dx.doi.org/10.5772/intechopen.93549*

The combustion characteristics of isolated celluloses were studied using thermogravimetric analysis (TGA). The analysis was done on a TGA (Q500, TA instrument). Samples of 6 mg (1.0) were first equilibrated at 25°C for 5 min and then heated at specific heating rates of 10, 15, 20, and 25°C/min to 900°C. The process was performed under constant nitrogen environment flowing at 20 mL/min. As the thermal decomposition progressed, the change in weight was recorded continuously as a function of temperature and time. **Figure 3** shows the isoconversion versus temperature at different heating rates for the isolated celluloses from DPW. The conversion curves for acid-base (colored) and Organosolv (black) methods below 300 and 340°C, respectively, showed similar thermal decomposition patterns at all heating rates. There was a slight shift toward higher temperature side with increasing heating rates, possibly due to the increasing thermal energy in the system [17]. However, at higher temperatures, the conversion pattern changed for both methods, possibly due to the change in the degradation chemistry of components under pyrolysis. It is worth to note that Organosolv cellulose showed better thermal stability than the acid-base cellulose. **Figure 4** shows the differential thermogravimetric (DTG) results against temperature at different heating rates for the DPW and the isolated celluloses. The results showed a typical thermal degradation of lignocellulose biomass. The curves of both samples moved downward as the heating

with uneven polished surface.

**Figure 2.**

**583**

*magnification X1000.*

**2.4 Thermogravimetric analysis (TGA)**

Organosolv cellulose (**Figure 2(c)**) showed an aggregate of cellulose block structure

#### **2.3 Scanning electron microscopy (SEM)**

The structural morphologies of the isolated cellulose were analyzed using the scanning electron microscope (JEOL Neoscope JCM-5000, Tokyo Japan). The samples were Au/C coated using vacuum spatter while clamped on the sample holder. The images were captured on spot size of 40 using 10 kV. The SEM results in **Figure 2** show a difference in the structural morphologies between the cellulose samples from the two methods. **Figure 2(a)** shows the original DPW with ring-like structures (see the arrow point) plausible to be the cellulose chiral nematic ordering, surrounded by irregular shaped structures which could be assumed to be lignin *Investigation of Nonisothermal Combustion Kinetics of Isolated Lignocellulosic Biomass… DOI: http://dx.doi.org/10.5772/intechopen.93549*

**Figure 2.**

indicating CdO, C]C and CdC bond stretching, between 2840 and 2926 cm1 indicating CdH stretching and 3200–3474 cm1 for OdH stretching, were assigned to cellulose, hemicellulose and lignin, respectively. Similar results were reported for other biomass in the literature [15]. However, there were noticeable changes in the functional groups of celluloses from the same biomass with reduction in the peak intensity, an indication of component(s) removal (plausibly lignin and hemicellulose). For instance, the would-be lignin and hemicellulose band intensity at 1037 cm1 greatly reduced an indication of component(s) removal. The CdH bond stretching in the region of 2840 assigned to lignin in DPW was absent in isolated cellulose samples. In addition, the reduced peak intensity between 845 and 1156 cm1 associated with the CdOdC asymmetrical stretching and glycosidic bond, a characteristic of cellulose, was observed. Furthermore, the decrease in OH vibration strength around 3200–3474 cm1 indicates a reduction in some of the OHcontaining compounds which are phenolics from lignin. It is worth to note that both extraction methods showed similar functional groups except that the Organosolv cellulose had CdH bond assigned to lignin in the region around 2326 and 2363 cm1 which was absent for the acid-base cellulose samples. The FTIR results showed the effectiveness of the cellulose isolation methods from DPW biomass complex. The samples were further characterized for their morphological differences using SEM

*FTIR spectrum of (a) lignocellulose DPW, (b) Organosolv cellulose and (c) acid-base cellulose.*

The structural morphologies of the isolated cellulose were analyzed using the scanning electron microscope (JEOL Neoscope JCM-5000, Tokyo Japan). The samples were Au/C coated using vacuum spatter while clamped on the sample holder. The images were captured on spot size of 40 using 10 kV. The SEM results in **Figure 2** show a difference in the structural morphologies between the cellulose samples from the two methods. **Figure 2(a)** shows the original DPW with ring-like structures (see the arrow point) plausible to be the cellulose chiral nematic ordering, surrounded by irregular shaped structures which could be assumed to be lignin

imaging technique.

**582**

**Figure 1.**

*Biotechnological Applications of Biomass*

**2.3 Scanning electron microscopy (SEM)**

*SEM images of the (a) original rachis, (b) acid-base cellulose and (c). Organosolv cellulose captured at magnification X1000.*

and hemicellulose. Acid-base cellulose in **Figure 2(b)** showed porous surface similar to those reported for cellulose from the teak wood [16]. However, Organosolv cellulose (**Figure 2(c)**) showed an aggregate of cellulose block structure with uneven polished surface.


**1. Introduction**

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by microorganisms and have very wide applicability as an alternative material for synthetic polymers, which cause environmental pollution as they are degraded under diverse earth environments, contrary to other biodegradable polymers [1,2]. Due to their excellent biocompatibility and in vivo degradation properties, their application is being extended to medical materials [3]. The developed PHA products are melt processed films [4,5] or foams [6] for environmentally friendly packing material and research is being carried out regarding their application to medical materials, such as biodegradable sutures from electrospinning [7] and scaffolds for tissue engineering from 3D printing [8], etc.

Poly(3-hydroxybutyrate) (P3HB) was the first biodegradable polymer to be studied among the PHAs, but had the limitation of having a narrow window for melt processing, as thermal degradation occurs very easily [9]. Furthermore, its crystallization rate is very low, resulting in large spherulites with the interface among these resulting in brittleness and the crystallization continually occurs during solidification, making the stable production of molded products difficult [10]. To overcome these problems, blending with biodegradable polymers that are compatible with P3HB, such as polylactic acid (PLA) and polybutylene succinate (PBS) [11,12], modification of the structure by branching or crosslinking

**Citation:** Zhang, T.; Jang, Y.; Lee, E.; Shin, S.; Kang, H.-J. Supercritical CO2 Foaming of Poly(3-hydroxybutyrate*co*-4-hydroxybutyrate). *Polymers* **2022**, *14*, 2018. https://doi.org/10.3390/ polym14102018

Academic Editor: Alberto Romero García

Received: 28 March 2022 Accepted: 10 May 2022 Published: 15 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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/).

through the addition of diverse additives [13,14], and the bacterial synthesis of copolymers from biodegradable monomers [15] have been suggested. The comonomers widely used with 3-hydroxybutyrate in the study of PHA copolymers are 3-hydroxyvalerate to prepare poly(3-hydroxybutyrate-*co*-3-hydroxyvalerate) [16], 4-hydroxybutyrate to prepare poly(3-hydroxybutyrate-*co*-4-hydroxybutyrate) [17], and hydroxyhexanoate to prepare poly(3-hydroxybutyrate-*co*-hydroxyhexanoate) [18].

Due to the higher price of PHAs compared with commodity polymers, they have been considered for use in high priced medical materials, but the decrease in production costs through mass production is resulting in higher possibilities of extending its application to packaging material [19,20]. As a relatively smaller amount of PHA is needed to prepare foamed material with a porous structure, the development of packaging material from PHA using foaming agents is being carried out [21]. Polymer foams can be made by the addition of diverse chemical foaming agents [22,23], using environmentally friendly supercritical fluid in melt extrusion [24,25], and the introduction of supercritical fluid to beads in the solid state to prepare foam beads [26]. Obtained foam beads can be molded by chest injection molding [27]. There are two typical processes to produce bead foam, continuous process and batch process using foam extrusion and autoclave, respectively. In the autoclave bead, polymer beads are saturated with a physical blowing agent, and pressure in the autoclave is suddenly released to expand the beads. Bead foaming is an appropriate processing method for semi-crystalline polymers, such as polypropylene (PP) [28], polyurethane (TPU) [29], and PLA [30]. The use of carbon dioxide supercritical fluid to maintain the environmentally friendly nature of biodegradable polymers is reported mostly in PLA foams [31–35], and in the case of PHA, the melt extrusion foaming of PHBV with supercritical CO2 is being carried out [36,37]. However, research on the foaming of P3HB has not been reported due to the lack of foamability. It is necessary to develop an eco-friendly supercritical CO2 process using biodegradable P(3HB-*co*-4HB) with improved chain flexibility.

The effect of 4HB content in the bead foam process using supercritical CO2 on the processing conditions and the resulting structure of the foams is studied. The effect of foaming temperature and pressure on the cell structure is studied in detail by evaluating the foam density, expansion ratio, etc. to explore the possibilities of application of these foams to packaging material. The effect of the addition of chain extending agents on the foam structure is also studied.

#### **2. Experimental**

The P(3HB-*co*-4HB) copolymer used in this study is a biodegradable polymer produced by CJ Cheiljedang, among which those with a 4HB content of 10–16% are confirmed to be crystalline and those with a 4HB content of 30 or 53.7% are confirmed to be non-crystalline. The chemical structure and 4HB content, along with molecular weights and heat of melting, are shown in Figure 1 and Table 1, respectively. A multifunctional styrene-acrylic oligomer (Joncryl, ADR 4370) purchased from BASF was used as a chain extender for the required control of the viscoelasticity of P(3HB-*co*-4HB) in the supercritical CO2 foaming process.

**Figure 1.** Chemical structure of P(3HB-*co*-4HB).

**Table 1.** Characteristics of P(3HB-co-4HB) used in this study.

P(3HB-*co*-4HB) of different 4HB contents were melt processed in an internal mixer (Haake, Rheomix600, Verden, Germany) at 20 rpm for 10 min at 140 ◦C, minimizing the thermal degradation to prepare samples of the same thermal history. When a 5 phr chain extender was added, the melt processing was carried out at 180 ◦C, which is the temperature at which the chain extender can react. The obtained P(3HB-*co*-4HB) samples were compression molded in a compression molding machine (QMESYS, QM900, Anyang, Korea) by heating at 140 ◦C for 2 min, raising the pressure to 8 MPa, and molded for 2 min, then quenched in water to obtain 1T thick 150 mm × 150 mm sheets. The sheets were cut into 5 mm × 5 mm pieces to use as beads for foaming.

The tan δ of P(3HB-*co*-4HB) was evaluated by measuring 1 mm × 2.5 mm sheets on a dynamic mechanical thermal analyzer (TA, DMA Q800/2980, New Castle, DE, USA) in the 30–140 ◦C range, at the oscillation frequency of 1 MHz.

The P(3HB-*co*-4HB) foam beads were prepared by adding 10 g of beads to a lab-made autoclave, as shown in Figure 2, diffusing supercritical CO2 into P(3HB-*co*-4HB) for 60 min at the supercritical condition of CO2 of 50–135 ◦C and 70–100 bar, then releasing the pressure abruptly to atmospheric pressure.

**Figure 2.** Schematic of supercritical CO2 assisted foaming process.

The structure of the P(3HB-*co*-4HB) foams was evaluated with SEM pictures, taken on a scanning electron microscope (Hitachi, SEM S-5200, Hitachi, Tokyo, Japan) and by measuring the density before foaming (ρ*p*) and the density after foaming (ρ*f*) from which the expansion ratio (Φ) was calculated according to the following Equation (1).

$$
\Phi = \frac{\rho\_p}{\rho\_f} \tag{1}
$$

Cell density (*Nf*) refers to the number of cells (*n*) per cubic centimeter (A) in a foamed sample and can be defined by the following Equation (2).

$$N\_f = \left(\frac{n}{A}\right)^{\frac{3}{2}} X \,\Phi\tag{2}$$

To measure the resilience rate of the crystalline P(3HB-*co*-10% 4HB) foam beads and noncrystalline P(3HB-*co*-30% 4HB) foam beads, the thickness of the foam beads was first determined as *T*0, then after compressing the foam beads to 50% its thickness and releasing the pressure after 30 min, the thickness was measured as *T*; the resilience rate was calculated according to the following Equation (3).

$$R = \frac{T}{T\_0} \times 100 \,\mathrm{(\%)}\tag{3}$$

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

#### *3.1. Foamability of P(3HB-co-4HB) Copolymer*

The SEM micrographs that show the possibilities of cell formation in the foams prepared in the range 50–130 ◦C at 90 bar for different 4HB contents are shown in Figure 3. As can be observed, the temperature at which the cells develop depends on the 4HB content. In the case of P(3HB-*co*-10% 4HB), whose relative crystallinity is highest, (23.97 J/g) as shown in Table 1, cell structure develops when processed at 130 ◦C, while in the case of P(3HB-*co*-16% 4HB), whose relatively crystallinity is lower (3.99 J/g), cell structure develops when processed at 100–110 ◦C. P(3HB-*co*-30% 4HB) is non-crystalline and the cell structure develops at a lower foaming temperature of 50–80 ◦C, but cell structure does not develop in the case of P(3HB-*co*-50% 4HB) in the range studied.

**Figure 3.** *Cont*.

Open cell Close cell

**Figure 3.** SEM micrographs of P(3HB-*co*-4HB) with different 4HB content foamed at 90 bar.

In supercritical foaming, the supercritical fluid diffuses into the polymer to create nuclei and they grow to form cells [38]. Nucleation and cell growth are affected by the crystalline/non-crystalline structure of the polymer and viscoelasticity, specifically, reasonable crystallinity and tan δ value under the foaming conditions are required for nucleation and continuous growth of the cell. In crystalline P(3HB-*co*-4HB), poor solubility and diffusivity of CO2 in crystallites caused the lowering of nucleation of the cell and prohibits cell growth, which resulted in less formability.

The change in tan δ with temperature for the P(3HB-*co*-4HB) is shown in Figure 4. The tan δ value and crystallinity are closely related to the chain mobility in the solid state. An increase in the tan δ value is observed with an increase in temperature and 4HB content. The tan δ is the ratio of loss modulus to storage modulus and shows a drastic change at various transition temperatures. The drastic change in the tan δ with temperature can be observed as the temperature reaches the melting transition temperature of crystalline P(3HB-*co*-4HB) or the rubber transition temperature of non-crystalline P(3HB-*co*-4HB). The drastic change occurs at around 130 ◦C and 110 ◦C for crystalline P(3HB-*co*-10% 4HB) and P(3HB-*co*-16% 4HB), respectively, as their melting temperature depends on the 4HB content. On the other hand, the change can be observed above 50 ◦C and 30 ◦C for noncrystalline P(3HB-*co*-30% 4HB) and P(3HB-*co*-50% 4HB), respectively, where the mobility of the non-crystalline chains start to increase. The tan δ value increases with 4HB content in P(3HB-*co*-4HB) and the change in the tan δ value also increases at the respective transition temperatures. In the case of the P(3HB-*co*-4HB) copolymers, the chain mobility of 4HB is greater than that of 3HB, resulting in a higher tan δ value and a more sensitive change with temperature. P(3HB-*co*-50% 4HB) exhibits a higher tan δ value than other P(3HB-*co*-4HB) copolymers regardless of the temperature, due to the too much chain flexibility of the 4HB chain. The adequate tan δ value required for cell growth in supercritical fluid foaming can be indirectly assessed by the tan δ value reflecting the polymer's viscoelasticity. As observed in Figure 3, for copolymers with a 4HB content up to 30%, the tan δ values below the transition temperature where the chains show a drastic change in their mobility are very similar, with the values being around 0.1. Therefore, foaming is expected to be possible below the transition where chain mobility is increased, which is 130 ◦C for P(3HB-*co*-10% 4HB), 110 ◦C for P(3HB-*co*-16% 4HB), and below 80 ◦C for P(3HB-*co*-30% 4HB). However, P(3HB-*co*-50% 4HB) exhibits a tan δ value above 0.3, regardless of the temperature reflecting the extremely chain mobility; thus, cell growth in the foaming process is not possible. As observed in Figure 3, in the case of P(3HB-*co*-10% 4HB) of low 4HB content, the temperature range where foaming is possible is very narrow around 130 ◦C, as the stiffness of the chains is high, resulting in a drastic decrease in the mobility of chains. In the case of P(3HB-*co*-16% 4HB) where the flexibility of the chains is increased

with an increase in 4HB content, foaming is possible in a relatively broader temperature range of 100–110 ◦C, and P(3HB-*co*-30% 4HB), which has the highest chain mobility, has the widest window where foaming is possible of 50–90 ◦C. Therefore, it is evident that in non-crystalline P(3HB-*co*-4HB) an adequate tan δ related to chain mobility above the glass transition temperature is an important factor in foaming.

**Figure 4.** DMA thermograms of various P(3HB-*co*-4HB) with and without ADR.

#### *3.2. Characteristics of P(3HB-co-4HB) Foams*

The cell structure of P(3HB-*co*-4HB) foams shown in Figure 3 generally shows thinner cell walls, which hinder the formation of closed cells and result in an open cell structure compared with thermoplastic polyurethane (TPU) foam [39], which is highly elastic, and the cell size is also very irregular. However, the non-crystalline P(3HB-*co*-30% 4HB) foam exhibits thicker walls and more regular size in the closed cell, compared with crystalline P(3HB-*co*-4HB). This can be explained by the crystallinity and their morphology affecting the foaming temperature. In crystalline P(3HB-*co*-4HB), the chain flexibility to foam the cell can be obtained just under the melting temperature, and then the strength for cell nucleation and growth is rapidly lowered with a further increase in temperature, which resulted in uncontrolled cell growth and cell coalescence. However, cell growth and stabilization in non-crystalline P(3HB-*co*-4HB) are more stable because the forming could be archived in a broad range of temperatures with high elastic properties above the glass transition temperature. The cell structure is related to the resiliency against applied pressure. Figure 5 shows the resilience rate of the crystalline P(3HB-*co*-10% 4HB) foam and noncrystalline P(3HB-*co-*30% 4HB) foam. The resilience of P(3HB-*co*-30% 4HB), which is noncrystalline due to the high 4HB content, is greater than that of crystalline P(3HB-*co*-10% 4HB). This is due to the higher elasticity of P(3HB-*co*-30% 4HB) from its higher 4HB content and also due to the thicker cell walls and more regular cell structure. The resilience rate of P(3HB-*co*-30% 4HB) foamed at different temperatures, as shown in Figure 5b and showed much higher resilience for foams prepared at lower temperatures. This shows that the elastic properties of P(3HB-*co*-30% 4HB), along with open/close cell structure, the thickness of the cell walls, and cell size, which in turn depends on an expansion ratio, has a close relationship with resiliency. These results suggest that the possibility of using crystalline P(3HB-*co*-4HB) foams as an environmentally friendly packaging material is higher than that of using it for industrial structural material, which requires elasticity and resiliency to recover to its original form after compression, as it has an open cell structure compared with TPU foams. On the other hand, the non-crystalline P(3HB-*co*-30% 4HB) foams prepared at low temperatures with 100% resilience rates suggest the possibility of developing it as an industrial structural material.

**Figure 5.** Resilience characteristics of P(3HB-*co*-4HB) foams; (**a**) crystalline P(3HB-*co*-16% 4HB); (**b**) non-crystalline P(3HB-*co*-30% 4HB).

Figure 6 shows the effect of foaming temperature on the density and the expansion rates calculated from density measurements before and after the expansion of P(3HB-*co*-4HB) beads of different 4HB content. The different foaming characteristic of P(3HB-*co*-30% 4HB) of different 4HB content was confirmed by the SEM micrographs in Figure 3. P(3HB*co*-50% 4HB) with the highest 4HB content does not show a significant change in density over the range studied, suggesting that cell growth from the diffused supercritical CO2 is not possible, due to its high tan δ value. The growth of cells in P(3HB-*co*-30% 4HB) at the foaming temperatures of 50–80 ◦C due to adequate tan δ and chain mobility can be confirmed. The cell growth of crystalline P(3HB-*co*-10% 4HB) and P(3HB-*co*-16% 4HB) at low temperatures is difficult due to the crystalline structure and lack of chain mobility, but near the melting temperature, chain mobility occurs and the tan δ becomes adequate for foaming to result in cell growth. The highest expansion ratios are obtained at 130 ◦C and 110 ◦C for P(3HB-*co*-10% 4HB) and P(3HB-*co*-16% 4HB), respectively. However, the uniformity of cells is very poor, due to the rapid decrease in strength to maintain cells during the stabilization step. The expansion ratio of non-crystalline P(3HB-*co*-30% 4HB) is below 6, while that of crystalline P(3HB-*co*-10% 4HB) and P(3HB-*co*-16% 4HB) is over 20. These P(3HB-*co*-4HB) foams have different expansion ratios, suggesting that they can be used to prepare foams for different applications.

**Figure 6.** Effect of foaming temperature on the structure of the P(3HB-*co*-4HB) bead foamed at 90 bar; (**a**) foam density; (**b**) expansion ratio.

The effect of foaming pressure on foam formation of P(3HB-*co*-4HB) is shown in Figure 7. The foaming temperature was set at the temperature at which the maximum expansion ratio could be obtained from the respective P(3HB-*co*-4HB) at 90 bar. The cell size increases with the increase in pressure. The foam density and expansion ratio of the P(3HB-*co*-4HB) copolymers shown in Figure 8 show a decrease in the foam density and an increase in the expansion ratio with an increase in pressure. An increase in the foaming pressure allows more diffusion of supercritical CO2 into P(3HB-*co*-4HB) to form a greater number of nuclei in P(3HB-*co*-4HB) beads, which results in a decrease in the foam density and an increase in the expansion ratio on cell growth when the pressure is relieved. This effect of pressure is more evident in the higher 4HB content P(3HB-*co*-4HB), showing that diffusion of the supercritical CO2 is easier in the non-crystalline P(3HB-*co*-4HB) than the crystalline P(3HB-*co*-4HB).

#### *3.3. Effect of Chain Extender on Foam Structures*

SEM micrographs that show the effect of the addition of a chain extender on the supercritical CO2 foaming are shown in Figure 9. Although the addition of a chain extender to crystalline P(3HB-*co*-10% 4HB) increases cell size when foamed at 130 ◦C, it does not affect the temperature window where foaming is possible, while in the case of P(3HB-*co*-16% 4HB), the temperature window appears at a higher temperature and is broadened from 110–100 ◦C to 120–130 ◦C. Figure 8b shows that although foaming of non-crystalline P(3HB-*co*-30% 4HB) is possible in the temperature range 50–90 ◦C in the absence of a chain extender, it is not when a chain extender is added. It was found that the degree of crystallinity of P(3HB-*co*-4HB) modified by a chain extender decreased dramatically, as shown in Table 1. This indicated that the crystalline structural change and the chain flexibility took place by chemical modification. As a result, the tan δ decreases observed in Figure 4 are caused by a structural change in the formation of chain branching or crosslinking from the reaction of epoxy groups of the chain extender Joncryl ADR 4370, containing epoxy groups, and the 3HB hydroxy groups in the crystalline P(3HB-*co*-4HB) of low 4HB content, resulting in a decrease in tan δ. However, in the case of P(3HB-*co*-4HB) of high 4HB content, viscosity decrease occurs instead of the aforementioned increase and decreases tan δ to decrease the foamability. Figure 10 shows the effect of a chain extender on the foam density and expansion ratio of the foam. The addition of the chain extender does not affect the foaming conditions in the case of P(3HB-*co*-10% 4HB) due to relatively high crystallinity, but a general increase in the expansion ratio with a decrease in foam density occurs. However, in the case of P(3HB-*co*-16% 4HB), the change in tan δ with the change in the chain structure allows foaming at temperatures above 110 ◦C and the foam density remains low above 110 ◦C to allow foaming at 120–130 ◦C, where foaming was not possible in the absence of a chain extender and an expansion ratio of around 20 can be obtained. Therefore, the control of the foaming properties with a chain extender is only possible in crystalline P(3HB-*co*-16% 4HB) with reasonably low crystallinity and tan δ value.

X300

**Figure 7.** SEM micrographs of P(3HB-*co*-4HB) foamed at various foaming pressure. Foaming temperatures of P(3HB-*co*-10% 4HB), P(3HB-*co*-16% 4HB), and P(3HB-*co*-30% 4HB) are 130 ◦C, 100 ◦C, 50 ◦C, respectively.

**Figure 8.** Effect of forming pressure on the structure of the P(3HB-*co*-4HB) foams. Foaming temperatures of P(3HB-*co*-10% 4HB), P(3HB-*co*-16% 4HB), and P(3HB-*co*-30% 4HB) are 130 ◦C, 100 ◦C, 50 ◦C, respectively; (**a**) expansion ratio; (**b**) cell size; (**c**) cell density.

**Figure 9.** SEM micrographs of foamed crystalline P(3HB-*co*-4HB) with and without chain extender.

**Figure 10.** Effect of chain extender on the structure of the P(3HB-*co*-4HB) foams; (**a**) foam density; (**b**) expansion ratio.

#### **4. Conclusions**

Biodegradable P(3HB-*co*-4HB) of different 4HB contents and foam beads processed with supercritical carbon dioxide was used to study the effects of 4HB content on the foaming conditions and foam properties. It was found that various foam structures for packaging and industrial materials were obtained by controlling the 4HB content in P(3HB*co*-4HB), which resulted in changes in the crystallinity and viscoelastic properties of P(3HB*co*-4HB). The low 4HB content P(3HB-*co*-4HB) that maintained a crystalline structure can be foamed just under the melting temperature, but the range of the foaming temperature becomes broader with an increase in the 4HB content. The expansion ratio is around 20 and an open cell structure is formed. The non-crystalline P(3HB-*co*-4HB) of high 4HB content has a relatively wide window of foaming around the temperature at which chain mobility occurs. The formed foam has thicker cell walls, expansion ratios of about 6, and the cell size is regular, resulting in enhanced resiliency against compression, compared with crystalline P(3HB-*co*-4HB). The chain extender changes the chain structure of crystalline P(3HB-*co*-16% 4HB), which changes the crystallinity and chain mobility and, thus, changes the temperature at which foaming is possible and the cell structure. To enhance the physical properties of P(3HB-*co*-4HB) foams, future studies for the blends and composites using P(3HB-*co*-4HB) may be considered.

**Author Contributions:** Conceptualization, H.-J.K.; investigation, T.Z., E.L. and Y.J.; writing—original draft, T.Z. and H.-J.K.; funding acquisition, E.L. and S.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** CJ Cheiljedang.

**Institutional Review Board Statement:** Not applicable.

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

**Data Availability Statement:** Data are in the authors' possession.

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


*3.4. Oral Health-Related Quality of Life*

Based on 45 edentulous patients, receiving an implant-supported overdenture, the OHIP-14 index reduced from 13.37/56 (SD 9.97) at baseline to 4.42/56 (SD 4.94) after three months of functional loading. This result in a large effect size of 0.90, suggesting a strong improvement in oral health related quality of life. Between 3 and 12 months, no further changes were observed, resulting in small effect size (0.04), indicative of a very stable result over time (Figure 7). The reduction was statistically significant for all seven domains after three months (Table 5). For functional limitation, physical disability and handicap the effect size was moderate. For the other four domains, a large effect size was observed and most expressed for physical pain with an effect size of 1.04. The latter is logically given the fact that improved denture retention results in less mucosal irritation and consequently fewer complaints related to pain suffering.

**Figure 7.** Boxplots representing the Oral Health Impact Profile-14 (OHIP-14) total score before surgery, 3 and 12 months after connection of the prosthesis with the implants. A score of 56 represents a maximal negative appreciation. ◦ Suspected outliers (between 1.5 × IQR and 3 × IQR above third quartile).

**Table 5.** Mean OHIP score and the mean difference for each of the seven domains before surgery and three months after connection with the calculated effect-size.

#### **4. Discussion**

The current paper focuses on implant treatment outcome in patients, which were completely edentulous in both jaws. Retention of the lower denture is a typical problem in this category of patients, especially in the mandible as compared to the maxillary denture. The denture in the mandible is less retentive because of a smaller crestal bone support, a more expressed degree of bone resorption, and unfavorable distribution of occluding forces, as well as additional pressure of the tongue yielding dislocating forces. Often this results in functional discomfort and pain, the latter because of the absence of keratinized mucosa. In the maxilla, the denture is supported on the crest and on the hard structure of the palate, which is covered by keratinized tissue. A vacuum present during mastication, between the palatal coverage of the denture and the underlying tissues, improves the retention. Consequently, fully edentulous patients have more complaints with mandibular dentures and an overdenture retained on two implants has therefore been suggested as of minimal care in order to provide functional comfort [6]. Implant treatment in denture wearing patients can be used for split mouth studies as was the case in the two clinical studies presented in the present paper. The focus was on implant type and surgical procedure, defined as implant survival, crestal bone loss and biologic peri-implant health. The latter is an important aspect because peri-implant diseases may jeopardize treatment outcome in the long run and are often related to aesthetic appreciation. Additionally, the patient-centered outcome was assessed by using a validated Oral Health Related Quality of Life questionnaire.

After three years of follow-up, no implant failures could be recorded in the present study and all remaining patients remained fully functional. This 100% implant survival is in line with current literature on implant overdenture therapy [47].

Initial bone remodeling is a healing phenomenon related to the surgical procedure mainly the exposure of bone and periosteum during implant placement, as well as the depth placement in the bone. Given the fact that implant survival with currently available dental implant systems is successful and quite predictable, the research focuses on implant success. Implant treatment is considered a success when high implant survival is combined with bone stability over time, because the latter reflects the health of the peri-implant tissues. Indeed, worldwide consensus defined that peri implantitis, a disease condition of the implant resulting in pocket formation between the implant and soft tissue, is always preceded by the bone loss [12]. Additionally, soft tissue health also affects the aesthetic outcome, especially in the partially edentulous patient. Although aesthetics was not the key issue in the present paper, the study conditions tested may provide clinical guidelines that do affect aesthetics, as well as peri-implant health outcomes.

In the present paper, minimal initial bone remodeling ranging from 0–0.7 mm was assessed. After the physiological initial bone remodeling, no further bone loss could be observed up to three years of function. The effect of soft tissue thickness and implant surface roughness on the crestal bone loss was evaluated. The applied split-mouth study design corrects for inter-individual variability from the estimates of the treatment effect [48]. The results showed that the initial bone remodeling was affected by the originally present soft tissue thickness, but not by the implant surface roughness. After implant

installation, a minimum of 3 mm soft tissue dimensions seems to be necessary for the re-establishment of the so-called "biologic width", indicative of the importance of the biologically guided implant placement. These findings are in accordance with an earlier published systematic review, including meta-analysis. There it is stated that implants placed with an initially thicker peri-implant soft tissue have less radiographic marginal bone loss in the short term [49]. Additionally, an increased early bone remodeling leads to implant surface exposure in patients with thin soft tissues, which increases the risk of on-going bone loss as shown by Vervaeke and colleagues in a nine year follow-up. A greater implant surface exposure increases the bacterial colonization of the implant surface, which could enlarge the chance to induce peri-implantitis [50]. From a clinical point of view, it is highly suggested that the surgeon adapts the surgical position of the implant in relation to the available pre-operative soft-tissue thickness.

It is generally accepted that osseointegration of moderately rough implants is enhanced as compared to minimally rough implants. This resulted in faster treatment protocols and reduced early failures. More recently, it was suggested that a minimally rough implant surface yields less crestal bone loss and less peri-implantitis on the long-term. A recent systematic review, including studies up to 10 years, reported on the survival rate and marginal bone loss of implants with different surface roughness. Implant survival was higher for moderately rough surfaces, but minimally rough surfaces showed the least marginal bone loss [51]. This outcome is in contrast to the outcome presented in another systematic review with meta-analysis. The latter evaluated the influence of the implant collar surface on marginal bone loss and revealed less bone loss for the rougher implant systems. However, 10 out of the 12 included studies showed results with less than five years of function. The only study with 10 years of follow-up showed less bone loss for the implants with a smooth collar compared to the implants with a rough collar. Yet, the authors stated that the results of their systematic review needed to be interpreted cautiously, due to several confounding factors [52]. Another systematic review with meta-analysis, which included only studies with at least, a five-year follow-up showed significantly less bone loss around smooth implant surfaces compared to moderately rough and rough implant surfaces [38]. Recently Donati and co-workers published the results of a 20-year follow-up RCT to evaluate the effect of a modified implant surface. In 51 patients at least one implant with a minimally rough surface and one with a modified surface was installed. The difference in mean bone level change between the two implant-systems was not statistically significant, and the moderate increase of implant surface roughness has no beneficial effect on long-term preservation of the peri-implant marginal bone level. A more detailed analysis of the paper revealed, however, that none of the 32 evaluated smooth implants showed more than 3 mm bone loss, whereas 3 out of the 32 modified implants showed bone loss between 3 and 6 mm. Only two smooth surface implants were diagnosed with peri-implantitis compared with five implants with a modified surface [53].

The findings of our paper are in accordance with the paper of Donati and co-workers, concluding that the surface roughness of the implant neck has no effect on bone level up to three years. The hybrid implant system used in our study combines the benefits of faster osseointegration, due to the moderately rough implant body, and the minimally rough surface around the implant neck suggests it is less prone to develop peri-implantitis [54]. Additionally, several studies conclude the beneficial effect of a smoother surface with a lower incidence of peri-implantitis and less bone loss on the long term. A further long-term follow-up of the current study population will elucidate the latter.

Besides implant survival and bone level stability, also peri-implant health is considered a perquisite for treatment success. Peri-implant health is defined on two levels. Plaque accumulation yields minor inflammation of the soft tissue surrounding the implant- restorative interface, coined as mucositis. It is diagnosed with bleeding of the tissues after probing the crevice between implant and mucosa. In a recent consensus report, the diagnosis of peri-implantitis has been redefined as a combination of probing pocket depths of at least 6 mm in combination with bleeding on probing or a bone level of at least 3 mm apical of the most coronal portion of the intraosseous part of the implant [12]. In our study,

no patients showed ongoing bone-loss in combination with bleeding and increasing probing pocket depths. Hence, the incidence of peri-implantitis was 0.0%.

The absence of peri-implantitis was found despite a high plaque level. This could be explained by the elderly, fully edentulous patient population. De Waal and colleagues revealed that edentulous patients restored with implants showed more plaque compared to partially edentulous patients restored with implants. However, the plaque in the fully edentulous patients harbours a potentially less pathogenic peri-implant micro-flora [55,56].

Another explanation for the relatively high plaque scores could be the dexterity problems inducing imperfect cleaning abilities in elderly patients. On the other hand, plaque is screened at a given moment in time during the clinical inspection and this may be several hours after cleaning and not necessarily reflects the overall hygiene of the patient over time.

This is the reason why the bleeding index is considered more useful. It reflects the degree of inflammation as a result of the long-term plaque control and is less momentarily. The current study revealed that high plaque score did not result in high bleeding scores.

The support of a mandibular overdenture by two implants has a significant positive effect on the quality of life. The OHIP-14 score was calculated irrespective of the implant group because it is a patient-related outcome variable. On all the seven domains measured with the OHIP-14 questionnaire a statistically significant difference was measured, all in favor of the support of a mandible overdenture by two implants. These findings are in accordance with a clinical trial reporting a significant improvement in satisfaction and health-related quality of life when subjects who received two implants are compared with subjects requesting a new conventional denture. Besides the improvement in the quality of life, they reported that patients requesting implants reported that tooth loss and denture wearing problems had a much greater impact in their quality of life than patients seeking conventional dentures [5].

#### **5. Conclusions**

Within the limitations of this study, it can be concluded that an implant supported mandibular overdenture significantly improves the quality of life, with limited biologic complications and a high survival rate of the implants. All seven domains of the OHIP-14 questionnaire significantly reduced when the mandible overdenture is supported by two implants. No differences were observed in crestal bone remodeling between minimally rough and moderately rough implant surfaces. However, initial bone remodeling was affected by initial soft tissue thickness. Anticipating biologic width re-establishment by adapting the vertical position of the implant in relation to the available soft tissue thickness may avoid peri-implant bone loss. The biologic variance of the patient might be more important compared to the configuration of the implant surface. Long-term follow-up of the study is necessary to determine the influence of early implant surface exposure and implant surface roughness on crestal bone loss, biologic parameters, mechanical complication, and implant survival.

**Author Contributions:** Conceptualization, R.D. and H.d.B.; methodology, R.D. and E.B.; software, R.D. and E.B.; validation, R.D., H.d.B. and E.B.; formal analysis, R.D., H.d.B. and E.B.; investigation, R.D., M.G., S.V. and C.M.; resources, R.D., H.d.B.; data curation, E.B.; writing—original draft preparation, R.D.; writing—review and editing, H.d.B., M.G. and S.V.; visualization, R.D.; supervision, H.d.B.; project administration, R.D.

**Acknowledgments:** Special thanks to Mario de Timmerman for the illustrations.

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

#### **References**

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*Article*