**4. Conclusions**

A new analytical methodology based on μ-MSPD-GC–MS/MS has been proposed for the first time for the simultaneous analysis of 14 multiclass organic UV filters in cosmetic and personal care products. The main parameters a ffecting μ-MSPD extraction have been optimized to obtain the highest extraction efficiency. Under the optimal conditions, which implies the use of Florisil as the dispersing agent and 1 mL of ACN as elution solvent, the method was successfully validated in terms of linearity, accuracy and precision. The proposed methodology was extended to other PCPs families, including fragrance allergens, preservatives, plasticizers and synthetic musks comprising a total of 78 compounds. Finally, to show the method suitability, it was applied to a broad range of real cosmetic samples present on the market, including sunscreen, make up, and hair-care products, among many others. In summary, the developed methodology provides a suitable, green, and fast tool to determine a broad range of cosmetic ingredients in a wide variety of cosmetic products, allowing simultaneous analysis of 78 compounds with very di fferent chemical nature in a single extraction and chromatographic run.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2297-8739/6/2/30/s1, Figure S1: Calibration plots for some representative compounds of each studied family, Table S1: Retention time and MS/MS transitions for the fragrance allergens, preservatives, plasticizers and synthetic musks.

**Author Contributions:** Conceptualization, M.L. and M.C.; methodology, M.L. and C.G.J.; validation, J.P.L., L.V. and M.L.; formal analysis, M.V., J.P.L., M.C. and L.V.; resources, M.L. and C.G.J.; writing—original draft preparation, M.L. and M.C.; writing—review and editing, M.C. and M.L.; supervision, M.L.; funding acquisition, M.L.

**Funding:** This research was supported by the project UNST10-1E-491 (Ministry of Science and Innovation, Spain). The authors belong to the Galician Competitive Research Group GPC2017/04 and to the CRETUS Strategic Partnership (ED431 2018/01). All these programs are co-funded by FEDER (EU).

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


# **Rule of Law and Human Mobility in the Age of the Global Compacts**

Edited by

Marion Panizzon, Daniela Vitiello and Tamas Molnar Printed Edition of the Special Issue Published in *Laws*

www.mdpi.com/journal/laws


**3. Proposed audio watermarking**

comprises of two main steps: (i) embedding method and (ii) extraction method. The secret key could be used for additional level of security. There are fundamentally three sorts of watermarking methods and are described here: (1) non-blind watermarking, (2) semi-blind and (3) blind watermarking. The process of watermarking that uses the original sound signal during the extraction procedure termed as "non-blind watermarking". The watermarking system uses a portion of the segment or some a part of the input audio signal then it is term as a "semi-blind watermarking". The watermarking system helps to retrieve the watermark without use of original audio signal or a part of an audio signal for extraction process termed

*Security and Privacy From a Legal, Ethical, and Technical Perspective*

as "blind watermarking" [14]. The paper covers proposed novel blind audio

sequence of binary bits which is inserted into the host signal. The audio watermarking scheme should have following basic characteristics: inaudibility, payload and robustness. **Figure 1** gives the visual representation of the requirements of data watermarking concept in digital audio, these three requirements

unsuccessful for an unauthorized user in any case.

and is provided by bits per second (bps).

forms the corners of the magic triangle.

lossy compression, etc.

the audio is being recorded.

(SNR).

**Figure 1.**

**198**

*Performance parameter magic triangle.*

watermarking scheme and its hardware implementation is performed in Xilinx ISE. The steps of algorithms are covered in Section 3. The watermark consists of a data

1.**Inaudibility:** The inserted data has to be "inaudible" in the watermarked digital music. Evaluation of the same is quantified using signal-to-noise ratio

2. **Security:** The algorithm should be secure where authorized person should able to only retrieve the watermark. The attempt of extracting watermark is to be

3. **Robustness:** The watermark should not be eliminated or removed by applying common processing techniques such as cropping, nonlinear and/or linear filter,

4.**Paylod (capacity):** It is defined as total information to be embedded in the host without having of any distortion. It is usually defined as the bit rate for the audio signal which is the actual number of bits inserted in the original audio

5. **Real-time processing:** The process of inserting into the original signal without much delay. It should be able to insert the watermark at same instance when

The proposed audio watermarking scheme is blind and robust and is based on DWT transformation. In the proposed scheme, only eight-audio samples of a single frame from two channels, is considered for watermarking process. The details of the embedded and extracted process are shown in the following section.


**3.1 Flue gas elements**

Sampling of flue gas elements from the entire boiler can be divided into three groups:

Co-Combustion of Coal and Alternative Fuels 71

chamber. The results of O2 concentration measurements for 60 % and 90 % nominal output (350 t/h steam) are illustrated in Fig. 7. The results of CO and NOX concentration

The measurment results suggest that there is intensive suppression of NOX formed increases in areas of secondary and tertiary air supply. CO concentration has developed as expected.

To determine the solid particle concentration in air flow, the Czech standard ČSN ISO 9096 needs to be observed. It is gravimetric determination of concentrations based on isokinetic sampling of solid particles from air flow. In the case of fluidized bed chambers the aim is to determine the solid particle concentration in the lower part of the fluidized bed layer and in the boiler furnace. For a bigger or smaller particle separation, the fluidized layer density varies depending on furnace height as well. The density in the lower part is in the range of 500 – 800 kg.m-3, in the upper part of the furnace with the circulating layer the range is 0.1- 0.5 kg.m-3. Pressure in the fluidized layer is always measured for various height levels. The

Fig. 7. Results of O2 concentration measurements by water-cooled sampling probe in relation to height at 60 % and 90 % nominal output during co-combustion tests in power

The concentration decreases if the secondary air supply is gradual.

**3.2 Solid particle concentration measurements**

station Tisová.

measurements for 60 % nominal output are illustrated in Fig. 8.

To monitor the fluidized bed boiler operation process O2, CO, CO2, NOX, and SO2 measurements can be taken. Other elements are usually monitored up to the exit from the separator of solid particles in front of the chimney. Fig. 5. illustrates a cooled sampling probe that might be used to take flue gases samples. The probe has an identical construction to that used for temperature measuring. During exhaustion gas is rapidly cooled down (from 800 oC to approx. 30 oC in cooled probe) so that there is no reaction with any other flammable waste gases. Gas is then sampled to be analyzed in the mobile laboratory. It is always recommended that a cooled probe is used to take samples from the furnace, cyclone and cyclone linking channels – Fig. 6. Sampling of flue gases from the second pass of the boiler occurs at temperatures safely below 800 oC. Thanks to that temperature a larger part of the gaseous sample is not able to oxidize quickly and thus it is possible to use a sampling tube made of stainless steel or sintered corundum, Al2O3. To set concentration (e.g., SO2), sampling channels must be heated up during the sampling operation so that no reaction with water occurs.

Fig. 5. Probe for flue gas sampling from fluid layer

Fig. 6. illustrates a sampling probe used in the detailed net measuring of O2 concentration in the boiler combustion chamber with a steam output 125 t/h. (15 % biofuels and 90 % lignite coal).

Fig. 6. Probe for flue gas sampling from furnace of boiler

A grid method of measuring O2 , CO and NOX concentration was used. Measurements were taken using instrumentation openings in the middle of the side walls of the combustion

To monitor the fluidized bed boiler operation process O2, CO, CO2, NOX, and SO2 measurements can be taken. Other elements are usually monitored up to the exit from the separator of solid particles in front of the chimney. Fig. 5. illustrates a cooled sampling probe that might be used to take flue gases samples. The probe has an identical construction to that used for temperature measuring. During exhaustion gas is rapidly cooled down (from 800 oC to approx. 30 oC in cooled probe) so that there is no reaction with any other flammable waste gases. Gas is then sampled to be analyzed in the mobile laboratory. It is always recommended that a cooled probe is used to take samples from the furnace, cyclone and cyclone linking channels – Fig. 6. Sampling of flue gases from the second pass of the boiler occurs at temperatures safely below 800 oC. Thanks to that temperature a larger part of the gaseous sample is not able to oxidize quickly and thus it is possible to use a sampling tube made of stainless steel or sintered corundum, Al2O3. To set concentration (e.g., SO2), sampling channels must be heated up during the sampling operation so that no reaction

Fig. 6. illustrates a sampling probe used in the detailed net measuring of O2 concentration in the boiler combustion chamber with a steam output 125 t/h. (15 % biofuels and 90 % lignite

A grid method of measuring O2 , CO and NOX concentration was used. Measurements were taken using instrumentation openings in the middle of the side walls of the combustion

with water occurs.

coal).

Fig. 5. Probe for flue gas sampling from fluid layer

Fig. 6. Probe for flue gas sampling from furnace of boiler

chamber. The results of O2 concentration measurements for 60 % and 90 % nominal output (350 t/h steam) are illustrated in Fig. 7. The results of CO and NOX concentration measurements for 60 % nominal output are illustrated in Fig. 8.

Fig. 7. Results of O2 concentration measurements by water-cooled sampling probe in relation to height at 60 % and 90 % nominal output during co-combustion tests in power station Tisová.

The measurment results suggest that there is intensive suppression of NOX formed increases in areas of secondary and tertiary air supply. CO concentration has developed as expected. The concentration decreases if the secondary air supply is gradual.


*Review* **The Oxidation Process and Methods for Improving Reactivity of Al**

**Deqi Wang, Guozhen Xu, Tianyu Tan, Shishuo Liu, Wei Dong, Fengsheng Li and Jie Liu \***

National Special Superfine Powder Engineering Research Center of China, School of Chemistry and Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China **\*** Correspondence<EMAIL_ADDRESS>Tel.: +86-025-8431-5042

**Abstract:** Aluminum (Al) has been widely used in micro-electromechanical systems (MEMS), polymer bonded explosives (PBXs) and solid propellants. Its typical core-shell structure (the inside active Al core and the external alumina (Al2O3) shell) determines its oxidation process, which is mainly influenced by oxidant diffusion, Al2O3 crystal transformation and melt-dispersion of the inside active Al. Consequently, the properties of Al can be controlled by changing these factors. Metastable intermixed composites (MICs), flake Al and nano Al can improve the properties of Al by increasing the diffusion efficiency of the oxidant. Fluorine, Titanium carbide (TiC), and alloy can crack the Al2O3 shell to improve the properties of Al. Furthermore, those materials with good thermal conductivity can increase the heat transferred to the internal active Al, which can also improve the reactivity of Al. Now, the integration of different modification methods is employed to further improve the properties of Al. With the ever-increasing demands on the performance of MEMS, PBXs and solid propellants, Al-based composite materials with high stability during storage and transportation, and high reactivity for usage will become a new research focus in the future.

**Keywords:** aluminum; oxidation process; reaction mechanism; modification method; development trend

#### **1. Introduction**

Aluminum (Al) is an important solid metal fuel, which has been widely used in connection between crystalline silicon [1], micro-electromechanical systems (MEMS) [2–5], polymer bonded explosives (PBXs) [6–12], and solid propellants [13–18] to provide energy. As a combustion agent, Al displays obvious advantages, such as low oxidant consumption, high combustion calorific value (31,070 J/g), and high measured specific impulse [19–21]. The dense and high melting point (about 2000 ◦C) alumina (Al2O3) shell will be formed when Al is exposed to air, which can improve the safety of aluminum powder in the process of production, storage, transportation and usage [22–24]. However, the oxidation process of Al is also closely related to the Al2O3 shell, which causes the inside active Al to become hindered (it cannot come into contact with oxidation components) [25–28]. As a result, the ignition and combustion reaction activity of Al is limited by the Al2O3 shell [21,29–31]. In addition, the combustion product Al2O3 will wrap onto the surface of the active Al again, which further hinders the combustion chemical reaction, resulting in an incomplete combustion of Al, and reduced energy release efficiency [29].

To solve the problem of the Al2O3 shell limiting oxidation activity and combustion efficiency, the oxidation process of Al has been studied extensively. To describe the ignition and combustion process of Al, some mechanisms, such as the oxidant diffusion mechanism [32–34], the Al2O3 crystal transformation mechanism [35–41], and the meltdispersion mechanism [42–46], have been proposed. Meanwhile, to improve the activity of Al, some new composite materials, such as Al-based metastable intermixed composites (MICs), flake Al-based composite materials, alloying and so on, have been found and used gradually [12,47–57].

**Citation:** Wang, D.; Xu, G.; Tan, T.; Liu, S.; Dong, W.; Li, F.; Liu, J. The Oxidation Process and Methods for Improving Reactivity of Al. *Crystals* **2022**, *12*, 1187. https://doi.org/ 10.3390/cryst12091187

Academic Editors: Rui Liu, Yushi Wen, Weiqiang Pang and Thomas M. Klapötke

Received: 4 July 2022 Accepted: 10 August 2022 Published: 24 August 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/).

A large number of reviews on Al have been published. Xiang Zhou [58] summarized the synthesis, ignition and combustion modeling, and applications of Al-based nanocomposites. Wei He [59] introduced the preparation and characterization of Al-based MICs. In addition, Xiaoxia Ma [60] focused on the preparation and fundamental properties of the Al-based core–shell structured nanoenergetic. However, they discussed the preparation and characterization methods, and hardly any reviews systematically summarized the relationship between the modification method and the different ignition mechanism of Al during the past decades. This review examines systematically the key influencing factors and the mechanisms during the Al oxidation process, and classified modification methods by the influencing factors of its transformation. Furthermore, it provides ideas for selecting modification methods of Al under different application conditions. What is more, the new trend of Al-based composite materials has been indicated.

#### **2. The Key Influencing Factors and the Reaction Mechanism during the Al Oxidation Process**

The Al particle is a typical core-shell structure with active Al wrapped in dense Al2O3. Therefore, the oxidation process of the Al particle is the evolution process of the oxidant, the alumina shell, and the inside active Al. Furthermore, the diffusion of the oxidant, the growth and rupture of the Al2O3 shell, and the melting and gasification of the inside active Al are the key influencing factors of the Al oxidation process.

#### *2.1. Effect of Diffusion of the Oxidant on the Al Oxidation Process*

The diffusion of the oxidant is a necessary process for Al oxidation. Zachariah [32–34] proposed a three-step oxidation process of Al (oxidant diffusion mechanism) on the basis of a well-known idealized "shrinking core" mechanism for spherical particles, which involves a reaction front moving radially inward separating an unreacted core with a completely reacted ash outer layer. As shown in Figure 1, the diffusion of the oxidant is influenced by three factors: the Al2O3 shell (generated on the surface in air), the Al2O3 ash (the product of oxidation reaction) and the chemical reaction of the oxidant with the inside active Al.

**Figure 1. The** three-step oxidation process of Al proposed by Zachariah.

Step 1: Diffusion of the oxidant through the Al2O3 shell to ash (Al2O3 generated by reaction, Al2O3 shell resistance).

Step 2: Diffusion of the oxidant through the ash (Al2O3 generated by reaction) to the surface of the inside active Al (ash resistance).

Step 3: Chemical reaction of the oxidant with the inside active Al at the unreacted core surface (chemical reaction resistance).

Since ash (Al2O3 generated by reaction) forms very rapidly, step 1 (Al2O3 shell resistance) can be ignored. This still leaves steps 2 and 3 from which to deduce the rate-limiting step. Then it is proved by experiments that the reaction of Al is controlled by diffusion. Therefore, the diffusion of the oxidant through the Al2O3 ash to the inside active Al is the rate-limiting step.

#### *2.2. Effect of the Al2O3 Shell on the Al Oxidation Process*

The Al2O3 shell on the surface of Al has a crucial effect on the oxidation process of Al. Trunov [35] analyzed the processes of simultaneous growth and phase transformations of Al2O3 during oxidation of the Al particle (Al2O3 crystal transformation mechanism). The process of Al oxidation can be divided into four stages, which can be seen in Figure 2. Firstly, amorphous Al2O3 on the Al surface gradually grows thicker and the reaction rate is controlled by the outward diffusion of Al cations [36]. Secondly, amorphous Al2O3 on the Al surface transforms to γ- Al2O3 when the critical thickness is approached, or when the temperature becomes sufficiently high [37–40]. Since the density of γ-Al2O3 exceeds that of the amorphous Al2O3, the Al2O3 shell on the Al surface ruptures. In addition, the inside active Al can come into contact with oxide, which greatly increases the reaction rate. With the growth and healing of the γ-Al2O3 shell, the reaction rate decreases significantly. Eventually, a regular polycrystalline layer of γ-Al2O3 forms by the end of the second stage. In stage three, the growth of γ-Al2O3 continues. In the meantime, γ-Al2O3 transforms the crystal phase to δ-Al2O3 or θ-Al2O3. Due to the density of δ-Al2O3, and θ-Al2O3 is similar to that of γ-Al2O3, the shell of Al2O3 will not rupture and the reaction will not change significantly. Additionally, the reaction rate is limited by the inward grain boundary diffusion of oxidant anions in stage three [36,41]. When the stable and denser α-Al2O3 forms by increased temperature, stage three ends. Stage four starts when Al2O3 is completely transformed to α-Al2O3. In stage three, the thickness of the γ-Al2O3 layer decreases, and the oxidation rate increases momentarily. Once most of the oxide layer is transformed to coarse and dense α-Al2O3, the contact between the internal active aluminum and the oxidized components is completely blocked, and the reaction rate decreases rapidly.

**Figure 2.** The change of the Al2O3 shell during the oxidation process of the Al particle, reprinted/adapted with permission from Ref. [35]. 2006, Dreizin, E. L.

#### *2.3. Effect of the Inside Active Al on the Al Oxidation Process*

Levitas [42–46] proposed a melt-dispersion mechanism when Al is heated rapidly. As shown in Figure 3, the volume change (6% volume expansion) due to the melting of the inside active Al can make the pressure of the Al2O3 shell reach about 11 GPa. That can cause spallation of the Al2O3 shell and the inside active Al can come into contact with the oxidant. Furthermore, the pressure inside the molten active Al will disperse the active Al into small droplets. Then the oxidation process of the active Al would occur rapidly.

**Figure 3.** The process of melt and dispersion of the active Al during the oxidation process of the Al particle, reprinted/adapted with permission from Ref. [46]. 2008, Levitas Valery I.

From the above discussion, it can be seen that the diffusion of the oxidant, the crystallization and the growth of the Al2O3 shell, and the melting and dispersion of the internal active Al, all have a crucial impact on the oxidation process of Al. The leading factors will change with the particle size and the heating rate of Al. Therefore, the oxidation process of Al under different conditions is not the same. At a slow heating rate, the diffusion of the oxidant plays a leading role, and the oxidation process of nano Al, which has a large specific surface area, conforms to the three-step oxidation process (proposed by Zachariah [32,33]). At the slow heating rate, the transformation and growth of the Al2O3 shell have the greatest influence on the oxidation process of micron Al, so the oxidation process of micron Al conforms to the Al2O3 shell transformation and growth mechanism (proposed by Trunov [35]). At a fast heating rate, the melting and dispersion of the internal active Al have the greatest influence on the oxidation process of nano Al, so the oxidation process of nano Al conforms to the melting diffusion mechanism (proposed by Levitas [42–46]).

The three crucial factors that determine the oxidation process of Al are: the diffusion of the oxidant, the transformation and growth of the Al2O3 shell, and the melting and dispersion of the internal active Al. As the key factors affecting the oxidation process of Al have been investigated, the oxidation process of Al under different conditions can be accurately described. Then the method of changing the Al oxidation process can be found, and the properties of Al can be improved.

#### *2.4. Effect of the Gas Phase Reaction on the Al Oxidation Process*

The gasification temperature of the internal active Al is lower than that of the Al2O3 shell, which leads to the gas phase reaction involving gas phase Al during Al oxidation [61]. As the models of the combustion process of micron Al, which were introduced both by Law [62] and Glassman [61], and later expanded upon by Brooks and Beckstead [63], the gas phase flame can drive the surface reactions of the Al particle. However, it is difficult to accurately characterize the process and products of gas phase reactions. Lynch [64,65] found that volatile suboxides existed during Al combustion, and Tappan [66] found the reaction between the gas phase Al and the Al2O3 shell. Although the gas phase reaction has been found, the reaction process under the different reaction conditions has not been accurately confirmed, and its effect on the oxidation of Al continues to be studied.

#### **3. Methods for Improving Al Properties**

To improve the properties of Al, the following methods have been investigated: metastable intermixed composites (MICs, reducing the diffusion of the oxidant), nano Al and flake Al (increasing the specific surface area of Al) for improving diffusion efficiency, fluorine modification [67], Titanium carbide (TiC) modification and alloying for cracking

the Al2O3 shell and the modification of good thermal conductivity materials (Ag, graphene) for improving heat transfer efficiency.

#### *3.1. Improving Diffusion Efficiency*

3.1.1. MICs

Metastable intermixed composites (MICs) are usually composed of a metal fuel and an oxidizer. The Los Alamos National Laboratory in the US was the first to study the combustion performance of MICs. Aumann [68] prepared the Al/MoO3 nano Al, which bulk energy density can reach 16 kJ/cm3, and the combustion rate is more than 1000 times that of the traditional thermit. The oxidizer of the Al-based MICs is usually metal oxide (bismuth oxide (Bi2O3), copper oxide (CuO), cuprous oxide (Cu2O), molybdenum oxide (MoO3), ferric oxide (Fe2O3), nickel oxide (NiO), tungsten trioxide (WO3)), halate (potassium perchlorate (KClO4), potassium periodate (KIO4), iron iodate (Fe(IO3)3), copper iodate (Cu(IO3)2), bismuth iodate (Bi(IO3)3)), and persulfide (potassium persulfate(K2S2O8)) etc. Al-based MICs effectively reduce the diffusion distance of oxidation, which has significant advantages in volume energy density, ignition, and burning rate [59]. Although, MICs have not been able to achieve efficient, safe and low cost batch preparation, and the reaction process of MICs is difficult to accurately control. Therefore, MICs are difficult to apply in solid propellants and PBXs these days [69].

Ludovic Glavier [70] prepared Al-based MICs by Al, Bi2O3, CuO and MoO3. Among them, as shown in Table 1, Al/Bi2O3 have the shorter the ignition delay time (5 μs), the highest burning rate (420 m/s), and the fastest pressurization rate (5762 kPa/μs). Egor A. Lebedev [71] prepared the MICs layer composed of Al, CuO and Cu2O by electrophoretic deposition, and the maximum heat release of MICs layer is 1954 J/g. Aifeng Jiang [72] prepared Fe2O3/nano Al by ball milling. The initial combustion temperature of Fe2O3/nano is about 600 ◦C. Ning Wang [73] prepared a Al@NiO core-shell structure composite microunit; the ignition temperature can be advanced to 531.5 ◦C, the heat release is 1410.2 J/g. Chunpei Yu [74] prepared 3D ordered macroporous Al/NiO MICs. Its heat releases up to 2462.27 J/g. Wei He [75] prepared Al/Energetic metal organic frameworks (EMOF) MICs, which can activate Al by eliminating Al2O3 shell and produce metal oxide by decomposing of EMOF. The heat release of Al/EMOF is 3464 J/g; the burning rate is 2.8 m/s.

**Table 1.** The thermal and combustion properties of Al-based MICs, reprinted/adapted with permission from Ref. [70]. 2015, Rossi Carole.

W. Lee Perry [76] prepared WO3·H2O/Al MICs, which had an energy release of approximately 1.8 MJ/kg at a rate of approximately 215 GW/m2. They found that the enhanced behavior of the hydrated MICs formulation resulted from the reaction of Al with the interstitially bound H2O, which had additional energy release and generated hydrogen gas.

Ahmed Fahd [77] compared and analyzed the thermal behavior of different nanothermite tertiary compositions based on nano Al, graphene oxide (GO), various salt and metallic oxidizers (as shown in Figure 4). The addition of GO enhances the reactivity of nanothermites with both salt and metallic oxidizers by reducing the reaction onset temperature, activation energy and increasing the heat release. For nanothermites with oxidizing salts, the heterogeneous solid–gas reaction mechanism plays a more important role than the condensed phase reactions. In general, nanothermites based on oxidizing salts are more reactive than those with metallic ones, as indicated in both theoretical and experimental data. Among them, the GO/Al/KClO4 nanothermite exhibits the highest

heat release (9614 J/g), while the GO/Al/K2S2O8 nanothermite shows the lowest onset temperature and activation energy (380 ◦C and 105 kJ/mol−1).

#### 3.1.2. Flake Al and Nano Al

Flake Al and nano Al have larger specific surface areas than that of sphere Al, which can improve the diffusion efficiency of oxidation [12,49–51,78–82]. However, the large-scale preparation process of nano Al and the effective Al content are still the key problems restricting its application.

Al has good ductility when it is subjected to external force, which would deform firstly. So flake Al is usually prepared by ball milling. As shown in Figure 5, in the process of ball milling, the sphere Al is extruded and sheared by ball milling beads. Under the action of force, sphere Al is changed to cake-like, firstly. In the second stage, the caked Al continues to be subjected to force and becomes flake Al; in the third stage, the flake Al would be broken and becomes smaller flake Al. So different thicknesses of flake Al can be prepared by controlling the parameters of ball milling.

**Figure 5.** The deformation process of Al during ball milling.

Lei Xiao [51] prepared flake Al powder by ball milling, which could increase the detonation heat of Al-containing explosives by 6.48%. William Wilson [49] found that the flake Al became small particles more easily by shock waves during combustion. Qingming Liu [78] found that the flake Al dust–air mixture could be ignited, and self-sustained detonation by an electric spark of 40 J. DeQi Wang [81] applied the flake Al powder to a solid propellant, which increased the burning rate of the propellant by 5.5%. A.L. Kuhl [50,82] found the flake-Al could increase the impulse of the TNT composite charges by the fast combustion of the flake-Al.

The main methods of preparing nano Al include the electric explosion of wires method [83] and the plasma-arc recondensation [84]. In order to protect the nano Al from oxidation during storage, it is usually necessary to form a coating film on the surface of the nano Al. Deluca L T [21,85] prepared a nano-coated Al with stearic acid (L-ALEX), palmitic acid (P-ALEX), and trihydroperfluoro-undecyl alcohol (F-ALEX), et al. (as shown in Figure 6).

**Figure 6.** The SEM image of P-ALEX (**a**) and F-ALEX (**b**), reprinted/adapted with permission from Ref. [21]. 2017, Deluca, L.T.

Nano Al is widely used in MICs and solid propellants, etc [86]. Deyun Tang [87] used tannic acid (TA) to coat on nano Al as an interfacial layer to bind with (Fe(IO3)3), copper iodate (Cu(IO3)2), bismuth iodate (Bi(IO3)3), respectively. For the energy release, Fe(IO3)3 based MICs can be increased to 24.1 kJ/cm3 (14.5% higher), whereas the Cu(IO3)2-based MICs to 22.8 kJ/cm3 (19.4%), and Bi(IO3)3-based MICs to 20.2 kJ/cm3 (3.1%). Deluca L [85] found that 100–200 nm Al can clearly increase the burning rate of the propellant.

Nano Al has high reactivity, which also produces some problems such as a high safety risk, a low content of active aluminum and sintering during combustion. Uncoated nano Al is classified by the International Air Transport Association as a highly flammable solid [88]. Weismiller [52] researched the effect of the particle size of Al and the oxidant on the properties of Al/CuO and Al/MoO3. As shown in Table 2, the properties of Al/CuO and Al/MoO3 are greatly improved due to the rapid decomposition of nano oxidants. However, due to the high Al2O3 content of nano Al, the nano Al does not show theoretical property advantages.

**Table 2.** Properties for Al/CuO and Al/MoO3, reprinted/adapted with permission from Ref. [52]. 2011, Weismiller, M. R.

Zachariah [89] found that the combustion product of nanothermites has two distinct populations of particles, as shown in Figure 7. The large particles include aluminum, oxidant, and reduced metal while the nano-sized particle is composed of reduced metal/metal oxide. As such large particles cannot be formed from the vapor phase condensation during the available transit time to the substrate, they must be formed in the condensed state as molten material.

**Figure 7.** The combustion product of nanothermites, reprinted/adapted with permission from Ref. [89]. 2015, Zachariah, Michael R.

#### *3.2. Cracking the Al2O3 Shell*

#### 3.2.1. Fluorine Modification

Fluoropolymers and fluorides can crack the Al2O3 shell of Al by the reaction between fluorine and Al2O3 shell. The curve of DSC will appear as a small exothermic peak before the main exothermic peak, caused by oxidation of Al. This phenomenon is known as preignition reaction (PIR), which is found by Osborne [90], and is verified by Zachariah by a quadrupole mass spectrometer and TG-DSC-MS coupling techniques [91]. The properties of the fluorine-modified Al are greatly improved due to the crack of the Al2O3 shell by PIR. Fluorine-modified Al can be used in MEMS and PBXs, but compatibility of fluoride with a propellant system is a key problem to realize its application in the propellant system.

Siva K. Valluri [92] prepared composite micro-units containing Al and NiF2 by reaction inhibition ball milling, which shortened the ignition delay time and improved the combustion efficiency of Al powder. Sergey Matveev [48] prepared Al/BiF3, which could produce 3200 K temperatures. Aifeng Jiang [72] prepared FeF3/Al@vinyltrimethoxysilane by ball milling, which had a large specific surface area (26.33 m2/g) and could be well-preserved from the air atmosphere and water. Additionally, the maximum heat release of FeF3/nano Al@vinyltrimethoxysilane can go up to 12,852 J/g.

Jena McCollum [93] composited Al with perfluoropolyethylene to advance the ignition temperature of Al to about 330 ◦C. Dong Won Kim [94] coated polyvinylidene fluoride (PVDF) onto the surface of Al (Al@PVDF), and the heat release of Al@PVDF at 900 ◦C was 11,040 J/g. Jun Wang from the Institute of Chemical Materials [95] prepared Carbon nanotubes/polytetrafluoroethylene (PTFE)/Al nanocomposites, which had a lower initial reaction temperature (reduced by 80 ◦C) and a shorter ignition delay time (reduced by 0.21 ms). Xiang Ke [96] coated PVDF onto the surface of Al to prepare reactive film materials, which appear PIR at 430 ◦C.

#### 3.2.2. TiC Modification

The reaction (as shown in Equation (1)) of Al2O3 and Titanium carbide (TiC) has been found during the preparation process of ceramics, which also can generate gas [97–99]. If this reaction can take place before the oxidation reaction of Al, the Al2O3 shell can be removed effectively, and the generated gas can also break the Al into small particles under high pressure. DeQi Wang [56] prepared the thick flake Al/TiC, which can crack the Al2O3 shell in-situ before 633 ◦C. As shown in Figure 8, the reaction of TiC with the Al2O3 shell on the Al surface to produce gas has been experimentally confirmed. The heat release of it can reach 21,419 J/g, and this powdered material has good application prospects in solid propellants and polymer bonded explosives (PBXs). This provides a new research idea

for solving the limitation of the Al2O3 shell on the Al oxidation process. However, the application of it in PBXs and the propellant system needs further exploration.

$$\rm Al\_2O\_3(s) + TiC(s) \to Al\_2O(g) + TiO(g) + CO(g) \tag{1}$$

**Figure 8.** The pores morphology of TF-Al/5%TiC surface at different temperature: 633 ◦C (**a**), 650 ◦C (**b**), 670 ◦C (**c**) and 690 ◦C (**d**), reprinted/adapted with permission from Ref. [56]. 2021, Jie Liu.

#### 3.2.3. Alloying

The oxide film on the surface of the Al-alloy particle may not be pure Al2O3 shell, which may have higher transmittance to oxidation. Therefore, the Al-alloy can be oxidized at lower temperatures. The oxidation product is not a dense structure like the Al2O3 shell. Accordingly, alloying is one of the effective ways to destroy the structure of the Al2O3 shell. Some metals (such as Li, Mg and Zr et al.) can be oxidized at a lower temperature than Al and can provide activation energy for the oxidation of Al, so that oxidation activity of Al-alloy is higher [53–55]. The preparation method of the high-density alloy is the key factor restricting the application of this modification method.

Hao Fu [55] prepared the Al- europium (Eu) alloy, which could be oxidized at 1065 ◦C and the heat release of the Al-Eu alloy is almost 5 times that of pure Al. Aobo Hu [53,54] prepared the Al-Zr alloy and the Al-W alloy. New alloy phases, ZrAl3, formed in the Al-Zr alloy, which changed the oxidation process (as shown in Figure 9a). Therefore, the Al-Zr alloy can complete combustion under high pressure. Furthermore, the Al-W alloy is almost completely oxidized in air at 1500 ◦C. In addition, as shown in Figure 9b,c, the gas product, WO3, increases the contact area between the active Al and the oxidant. As a result, the properties of the Al-W alloy have been improved greatly. Fahad Noor [100] prepared the Al-Cu alloy, which had the lower ignition temperature (565 ◦C).

#### *3.3. Accelerating the Melting of the inside Active Al*

The composite made of high thermal conductivity material and Al can increase the rate of Al absorbing heat, which can accelerate the melting of active Al inside and break through the limitation of Al2O3 shell. Consequently, the inside active Al can make contact with the oxidant and the reactivity of Al will be improved. However, as with MICs, how to achieve an efficient, safe and low cost batch preparation is the key problem when it is used in PBXs and propellants.

Jinpeng Shen [101] investigated the effects of nano-Ag on the combustion wave behavior of Al/CuO. The experimental observations confirm that the presence of nano-Ag particles improves the heat transfer efficiency, and the first exothermic peak temperature decreases from 607.8 ◦C to 567.6 ◦C.

Ahmed Fahd [77] compared and analyzed the thermal behavior of different nanothermite tertiary compositions based on Al, graphene oxide (GO), and various salt and metallic oxidizers. The addition of GO enhances the reactivity of nanothermites with both salt and metallic oxidizers by reducing the reaction onset temperature, activation energy and increasing the heat release.

#### **4. New Trend of Improving Reactivity of Aluminum Powder**

With the deepening of research on the Al oxidation process, various factors restricting Al oxidation have been found, gradually. The methods of improving the reactivity of Al have been integrated, which can simultaneously change multiple conditions

in the oxidation process of aluminum powder to improve the properties of Al from multiple perspectives.

Jena McCollum [102] investigated the reactivity of Al/MoO3@perfluoropolyethers (PFPE) and Al/CuO@PFPE. As shown in Table 3, fluorine−Al-based surface reaction can improve the reactivity of Al/MoO3. However, the reactivity of CuO reduces when the PFPE concentration is increased. Lei Xiao [47] successfully assembled Al/CuO/PVDF/RDX, and the combustion properties of microspheres is mainly affected by the content of RDX. Aifeng Jiang [72] prepared Fe2O3/nano Al and FeF3/nano Al@vinyltrimethoxysilane by ball milling. The initial combustion temperature of Fe2O3/nano is about 600 ◦C. FeF3/nano Al@vinyltrimethoxysilane had a large specific surface area (26.33 m2/g) and could be well-preserved from air atmosphere and water. Furthermore, the maximum heat release of FeF3/nano Al@vinyltrimethoxysilane can go up to 12,852 J/g.

**Table 3.** The data DSC of Al/MoO3@PFPE and Al/CuO@PFPE, reprinted/adapted with permission from Ref. [102]. 2015, Michelle L. Pantoya.

In the meantime, the safety of Al during storage, transport and usage also should be addressed when improving the properties of Al. The design and preparation of Al composites with high stability during storage and transportation, and high reactivity during usage, will become a research focus in the future.

DeQi Wang [56] designed flake Al/TiC, with TiC embedded on the surface of flake Al, which retains the Al2O3 shell to keep Al stable during storage and transportation, and cracks the Al2O3 shell to improve the reaction activity and combustion efficiency by the reaction of Al2O3 and TiC before 633 ◦C. In addition, the heat release of flake Al/TiC is 21,419 J/g at 985.6 ◦C.

#### **5. Summary and Prospect**

With increasing research, the oxidation process of Al has been gradually revealed, which is closely related to the reaction conditions. The oxidation process of nano Al (large specific surface area) is mainly restricted by oxidant diffusion under a slow heating rate, and conforms to a three-step oxidation process, proposed by Zachariah. The Al2O3 shell has the greatest influence on the micron Al oxidation process and Al2O3 shell transformation, and the growth mechanism can well explain this process, which was proposed by Trunov under the slow heating rate. The melting and dispersion of the internal active Al has a great impact on the oxidation process of nano Al under a fast heating rate, which can be described by the melting diffusion mechanism proposed by Levitas.

Therefore, to improve the properties of Al, the key influencing factors of the Al oxidation process need to be changed. MICs, flake Al and nano Al can improve the diffusion efficiency of the oxidant during Al oxidation, which can influence the properties of Al under a slow heating rate. Since the Al2O3 shell can be cracked by fluorine, TiC, and alloy, the properties of Al under a fast heating rate can be changed by them. Those materials with good thermal conductivity can increase the heat transferred to the internal

active Al, so the properties of Al under a fast heating rate can be improved by good thermal conductivity materials modification.

In order to improve the properties of Al more comprehensively, an integration of different modification methods has been employed, such as fluoride coated nano Al-based MICs, flake Al/TiC, various material-covered Al and so on. Furthermore, the safety of Al during storage, transport and usage also needs to be addressed when improving the properties of Al. The design and preparation of Al composites with high stability during storage and transportation, and high reactivity during usage, will become a research focus in the future.

**Author Contributions:** Conceptualization, D.W.; formal analysis, D.W.; investigation, G.X. and W.D.; writing—original draft preparation, T.T.; writing—review and editing, S.L.; supervision, F.L.; project administration, J.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** Not applicable.

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

#### **References**


*2.6. Validation*

Finally, to evaluate the effectiveness of the classification process, a statistical validation is applied, based on nine metrics: true positive (TP) (conditions correctly classified), true negative (TN) (controls correctly classified), false positive (FP) (controls incorrectly classified), false negative (FN) (conditions incorrectly classified), sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy.

Sensitivity can be calculated by Equation (4),

$$Sensitivity = \frac{TP}{TP + FN'} \tag{4}$$

describing the true positive rate, i.e. the probability that a depressive episode is classified rightly.

Specificity can be calculated by Equation (5),

$$Specificity = \frac{TN}{FN + TP'} \tag{5}$$

describing the true negative rate, i.e. the probability that a non-depressive episode is classified rightly.

The PPV value can be defined by Equation (6), being the probability that a new episode of a person suffering from depression is classified as a depressive episode

$$PPV = \frac{Sensitivity \* Prevalence}{Sensitivity \* Prevalence + (1 - Specificity) \* (1 - Prevalence)},\tag{6}$$

where *Prevalence* is the percentage of observations with a condition, in this case depressive episodes.

The NPV value can be defined by Equation (7), being the probability of a episode with absence of depression is classified as negative.

$$NPV = \frac{Specificity \ast (1 - Prevalence)}{(1 - Sensitivity) \ast Prevalence + Specificity \ast (1 - Prevalence)},\tag{7}$$

*Diagnostics* **2020**, *10*, 162

Finally, the accuracy can be calculated with Equation (8), being the total probability that one episode is classified correctly.

$$Accuracy = \frac{TruePositive + TrueNegative}{TruePositive + TrueNegative + FalsePositive + FalseNegative}^{\prime} \tag{8}$$

## **3. Experiments and Results**

In Figure 2 is presented a comparison of the motor activity between a control and a condition in different hours of the day. In the differences can be observed that every hour activity of control and condition shows different patterns. From these data, a segmentation is applied to form data intervals containing the information of one hour time lapses. The structure of the data for every observation is contained by 61 columns; one column for the monitored hour and one column for each minute (60 columns) of motor activity. This segmentation allowed the classification of depressive episodes per hour.

**Figure 2.** Comparison of motor activity in different hours of the day between a control an a condition.

From Figure 2 they can also be distinguished different patterns on the activity of control and condition subjects in different moments of the day. In Figure 2a, at 5 p.m., the control subject signal collected presents higher levels of activity in contrast with the depressive patient. This can be the most expected conduct of a patient with depression. Nevertheless, as the day ends the signal of both, control and condition, starts to change as shown in Figure 2b–d, depressive activity containing higher values.

Therefore, based on this, the data is treated in three different sets, each one corresponding to different moments of the day. One set corresponding the day, one to the night and one to the full day.

Then, for the next stage a feature selection is proposed. The results for each dataset are shown in Table 4. Accuracy is the metric used to evaluate the performance of the models constructed by the FS approach including different number of features (two, four, five, six, seven, eight, nine and ten).

In case of Night Data and Full Day Data datasets, higher accuracy is achieved with nine-features model in classification of depressive and non-depressive episodes . Day data best model is comprised by 8 features, however, the difference with nine-features model is less than 0.1 percent.

**Table 4.** Forward Selection results for the night, day and full day data.

After the feature selection with FS approach, validation step is done in two steps. Firstly, classification is done with the best nine-features model for each dataset, mentioned in Table 5, even when Day Data has higher accuracy with 8 features, best nine features is selected to compare the performance in same circunstances with the other two datasets. The results of this classification are shown in Table 6, described as Best 9 Features Day, Best 9 Features Full Day and Best 9 Features Night. In addition to accuracy, which was used in FS step, sensitivity and specificity were calculated in this validation to give a wide view of the performance of the models.

Secondly, classification is performed using the nine-features set of the Best 9 Features Night, applied to the Day Data and the Full Day Data datasets, because this nine features model is the one which outperforms all other nine features models in all proposed metrics. This in order to evaluate the performance of a general model, i.e. a unique model for all the time of the day. The results of this classification are described as the Best Model Day, Best Model Full Day in Table 6.

**Table 5.** Best nine features model in every dataset; day, night and full day.

From this table can be observed that every model has a significant performance in the classification of depressive episodes. Sensitivity values oscillate from 98.24% to 99.37% and specificity range oscillates between 98.08% and 99.31%, establishing an almost perfect classification of depressive and non-depressive episodes.

The lowest, but still being good results, are those from the Day Data with nine features selected from the Night Data.

**Table 6.** RF results for datasets using the nine features models and the best model from the forward selection on night dataset.


*2.4. Analysis of the Genome Locus Encoding the 7-Hydroxycoumarin-Inducible Protein*

Analysis of *Pseudomonas* sp. 7HK4 genome sequences (35 contigs) showed that the inducible 31.2 kDa protein belongs to the fumarylacetoacetate (FAA) hydrolase family, which includes such enzymes as 2-keto-4-pentenoate hydratase, 2-oxohepta-3-ene-1,7-dioic acid hydratase, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase, or bifunctional isomerases/decarboxylases (catechol pathway), as shown in Figure S3 in the Supplementary Material. The FAA family proteins are usually involved in the last stages of bacterial metabolism of aromatic compounds [17–19], suggesting that 31.2 kDa protein from *Pseudomonas* sp. 7HK4 participates in the final steps of 7-hydroxycoumarin metabolism, after oxidative cleavage of the aromatic ring.

Adjacent to the 31.2 kDa protein-encoding gene, two open reading frames (ORFs) were identified. All three genes are arranged on the same DNA strand, and are separated by short intergenic regions, suggesting that these genes are organized into an operon, as shown in Figure 3B. The putative operon was designated *hcdABC* (**h**ydroxy**c**oumarin **d**egrading operon), where *hcdC* encodes the 31.2 kDa protein. A BLAST analysis of *hcdA* and *hcdB* sequences revealed that these genes encode the putative FAD-binding hydroxylase and ring-cleavage dioxygenase, respectively. HcdA protein was not assigned to any family, but it showed similarity to a putative 2-polyprenyl-6-methoxyphenol hydroxylase, as shown in Figure S3 in the Supplementary Material. This type of enzyme belongs to class A of FAD-binding monooxygenases, which are involved in bacterial degradation of aromatic compounds [20–23]. The product of the hcdB gene belongs to the cl14632 superfamily that combines a variety of structurally related metalloproteins, including the type I extradiol dioxygenases, as shown in Figure S3 in the Supplementary Material. The type I extradiol dioxygenases catalyze the incorporation of both atoms of molecular oxygen into aromatic substrates that results in the cleavage of the aromatic rings [24,25].

## *2.5. Expression and Substrate Specificity of HcdA Hydroxylase*

For further characterization of HcdA hydroxylase, the *hcdA* gene was amplified by PCR and cloned into the pET21b expression vector. The sequence was confirmed by Sanger sequencing. The recombinant C-terminally His6-tagged protein was produced in *Escherichia coli* BL21, and purified by affinity chromatography. The purified enzyme migrated as a ~62 kDa band on SDS-PAGE, as shown in Figure S4A in the Supplementary Material, and had a bright yellow color with absorbance maxima at 380 and 450 nm wavelengths, as shown in Figure S4B in the Supplementary Material, suggesting that the protein contains a tightly bound flavin [26–28]. The gel-filtration showed that the purified HcdA protein is a monomer, as shown in Figure S4C in the Supplementary Material. The specificity for both flavin and nicotinamide cofactors was investigated. The HcdA hydroxylase was able to utilize either NADH or NADPH, although the oxidation rates of NADPH were almost 2-fold lower. Kinetic characterization of HcdA protein is presented in the Supplementary Material.

The activity of the HcdA enzyme was assayed in the presence of NADH cofactor against various substrates. The highest rate of oxidation of NADH was recorded in the presence of 3-(2,4-dihydroxyphenyl)-propionic acid. A 40-fold lower rate was observed when *trans*-2,4-dihydroxycinnamic acid was used as the substrate. The HcdA was not active towards *trans*-cinnamic, *cis*-2,4-dihydroxycinnamic, 3-(2-hydroxyphenyl)-propionic, 3-(2-hydroxyphenyl)-2-propenoic, 3-(4-hydroxyphenyl)-2-propenoic, 3-(3-hydroxyphenyl)-2-propenoic, 3-(2-bromophenyl)-propionic, 3-(2-nitrophenyl)-propionic, and 3-phenylpropionic acids, cinnamyl alcohol, pyrocatechol, 3-methylcatechol, 4-methylcatechol, 2-propylphenol, 2-propenylphenol, 2-ethylphenol, o-cresol, o-tyrosine, resorcinol, 2,3-dihydroxypyridine, 2-hydroxy-4-aminopyridine, N-methyl-2-pyridone, N-ethyl-2-pyridone, N-propyl-2-pyridone, N-butyl-2-pyridone, indoline, and indole. These data demonstrate that the HcdA is a highly specific monooxygenase, which shows a strong preference to 3-(2,4-dihydroxyphenyl)-propionic acid. The addition of His6-tag did not affect the enzymatic activity of the HcdA protein.

The product of the reaction catalyzed by the HcdA hydroxylase was analyzed by UV-VIS absorption spectroscopy and HPLC-MS. A new UV absorption maximum was observed at 340 and 490 nm upon addition of 3-(2,4-dihydroxyphenyl)-propionic acid to the reaction mixture. The consequent red coloring was observed, which indicated the presence of *para*- or *ortho*-quinone [29], a presumed product of the autooxidation of the corresponding hydroquinone. The same coloration was also observed in vivo when *Pseudomonas* sp. 7HK4 cells were grown in an excess of 7-hydroxycoumarin and when *Escherichia coli* BL21 cells harboring the p4pmPmo plasmid were cultured in the presence of 3-(2,4-dihydroxyphenyl)-propionic acid. HPLC-MS analysis of the reaction mixtures of both in vitro and in vivo bioconversions confirmed the formation of 3-(trihydroxyphenyl)-propionic acid and quinone, found [M−H]− masses were 197.00 (traces seen only in vivo) and 195.00, respectively, as shown in Figure 4. However, the structure of the product was not confirmed at this stage by chemical analysis since it was not possible to chromatographically separate the product from the reaction mixture. The substrate and product have similar structures and chemical properties; therefore, both were detected under the same HPLC-MS chromatogram peak with retention time of ~5.5 min.

**Figure 4.** HPLC-MS analysis of 3-(2,4-dihydroxyphenyl)-propionic acid bioconversion mixture in vitro. UV 254 nm trace of 3-(2,4-dihydroxyphenyl)-propionic acid and its hydroxylated product under the same peak with retention time 5.472 min (on the left), and MS spectrum of the dominant peak (on the right). The negative ions [M−H]− generated are at *m*/*z* 181 (3-(2,4-dihydroxyphenyl)-propionic acid), 195 (product of 3-(trihydroxyphenyl)-propionic acid autooxidation), and 363 (dimer of 3-(2,4-dihydroxyphenyl)-propionic acid).


6.3.1. Scanning electron microscope (SEM)

The two-dimensional morphology of MSCs demonstrated by scanning electron microscope (SEM) [38] showed the spindle-shaped cells with eccentric nuclei and several thin cytoplasmic processes extending from the edge of the cell surface in P5 and P9. In addition, cells in P 9 maintained their spindle shape (Figure 4). These SEM results were also reported [49].

Figure 2. Cultured human bone marrow derived stromal cell from passage 5, showing adherent cells with their characteristic spindle shape (arrow) [38]. Scale bar 200 μm.

Figure 3. Cultured human bone marrow derived stromal cell from passage 9, showing adherent cells with their characteristic spindle shape (arrow) [38]. Scale bar 200 μm.

#### 6.3.2. Transmission electron microscope (TEM)

Electron microscopic examination of MSCs in culture revealed the presence of euchromatic nucleus associated with abundant cell organelles which are considered as an indicator of an active cell (Figure 5). The spindle-shaped cells showed large irregular, euchromatic nucleus and the peripheral heterochromatin was slightly condensed along the inner surface of the nuclear membrane and nuclear pores (Figure 6). The cytoplasm showed many elongated profiles of rough endoplasmic reticulum and multiple mitochondria (Figure 7). Cytoskeletal structures were seen as fine filaments running parallel to the long axis of the cell near the nuclear membrane as well as beneath the cell membrane (Figure 6).

The same features of active MSCs were noticed after 14 days in culture. The cells exhibited a large euchromatic nucleus with numerous profiles of rough endoplasmic reticulum and multiple rounded mitochondria. In addition, the cell surface showed thin pseudopodia (Figure 7). Cytoskeletal filaments were irregularly dispersed in the cytoplasm as well as around the nucleus (Figure 7). Such observation was explained by the fact that the intracellular organelles architecture is organized by the cytoskeleton [36, 50, 51].

Figure 4. Cultured human bone marrow derived stromal cell, showing spindle shape cell with an eccentric nucleus (N) and multiple processes (P) [38]. Scale bar 50 μm.

> Moreover, after 21 days in culture, the cells showed clearly demarcated nucleolus (Figure 8). In addition, numerous large macro vesicles associated with the mature face of the Golgi complex were clearly depicted (Figure 9). These cells are now ready for differentiation once in the appropriate media. The structure of these cells would differ during the process of differentia-

> Figure 7. Transmission electron micrograph of cultured human bone marrow derived stromal cell on day 14. The cytoplasm exhibits numerous profiles of rER, mitochondria (M), and well-developed Golgi complex (G). The cell membrane exhibits a pseudopodium (Pd). Cytoskeletal filaments are irregularly dispersed in the cytoplasm (arrows). Part of

> Figure 6. Transmission electron micrograph of cultured human bone marrow derived stromal cell showing part of the same cell exhibiting an euchromatic nucleus (N) with nuclear pores (arrow heads). The peripheral heterochromatin (H) is seen along the inner aspect of the nuclear membrane. Fine cytoskeletal filaments are noticed parallel to the long axis of the

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35

http://dx.doi.org/10.5772/intechopen.76870

cell near the nuclear and cell membranes (arrows) [38]. Scale bar 0.5 μm.

an euchromatic nucleus is also seen (N) [38]. Scale bar 0.5 μm.

Another ultrastructure feature of MSCs is the presence of vesicles in the cytoplasm. Intercellular communication can be mediated through direct cell–cell contact or transfer of secreted molecules. Recently, a third mechanism has emerged that involves intercellular transfer of extracellular vesicles. Cells release into the extracellular environment membrane vesicles either of endosomal origin or of plasma membrane origin. They are named exosomes and microvesicles, respectively [52]. In the study [38] carried out on isolated MSCs, showed vesicular trafficking. (Figure 10) These vesicles were prominent after the cells were cultured in a

tion accordingly.

Figure 5. Transmission electron micrograph of cultured human bone marrow derived stromal cell on day 7. The cell is spindle in shape with an euchromatic nucleus (N). The cytoplasm shows mitochondria (M) and multiple lysosomes (L) [38]. Scale bar 1 μm.

6.3.2. Transmission electron microscope (TEM)

34 Stromal Cells - Structure, Function, and Therapeutic Implications

nuclear membrane as well as beneath the cell membrane (Figure 6).

architecture is organized by the cytoskeleton [36, 50, 51].

and multiple processes (P) [38]. Scale bar 50 μm.

[38]. Scale bar 1 μm.

Electron microscopic examination of MSCs in culture revealed the presence of euchromatic nucleus associated with abundant cell organelles which are considered as an indicator of an active cell (Figure 5). The spindle-shaped cells showed large irregular, euchromatic nucleus and the peripheral heterochromatin was slightly condensed along the inner surface of the nuclear membrane and nuclear pores (Figure 6). The cytoplasm showed many elongated profiles of rough endoplasmic reticulum and multiple mitochondria (Figure 7). Cytoskeletal structures were seen as fine filaments running parallel to the long axis of the cell near the

The same features of active MSCs were noticed after 14 days in culture. The cells exhibited a large euchromatic nucleus with numerous profiles of rough endoplasmic reticulum and multiple rounded mitochondria. In addition, the cell surface showed thin pseudopodia (Figure 7). Cytoskeletal filaments were irregularly dispersed in the cytoplasm as well as around the nucleus (Figure 7). Such observation was explained by the fact that the intracellular organelles

Figure 4. Cultured human bone marrow derived stromal cell, showing spindle shape cell with an eccentric nucleus (N)

Figure 5. Transmission electron micrograph of cultured human bone marrow derived stromal cell on day 7. The cell is spindle in shape with an euchromatic nucleus (N). The cytoplasm shows mitochondria (M) and multiple lysosomes (L) Figure 6. Transmission electron micrograph of cultured human bone marrow derived stromal cell showing part of the same cell exhibiting an euchromatic nucleus (N) with nuclear pores (arrow heads). The peripheral heterochromatin (H) is seen along the inner aspect of the nuclear membrane. Fine cytoskeletal filaments are noticed parallel to the long axis of the cell near the nuclear and cell membranes (arrows) [38]. Scale bar 0.5 μm.

Figure 7. Transmission electron micrograph of cultured human bone marrow derived stromal cell on day 14. The cytoplasm exhibits numerous profiles of rER, mitochondria (M), and well-developed Golgi complex (G). The cell membrane exhibits a pseudopodium (Pd). Cytoskeletal filaments are irregularly dispersed in the cytoplasm (arrows). Part of an euchromatic nucleus is also seen (N) [38]. Scale bar 0.5 μm.

Moreover, after 21 days in culture, the cells showed clearly demarcated nucleolus (Figure 8). In addition, numerous large macro vesicles associated with the mature face of the Golgi complex were clearly depicted (Figure 9). These cells are now ready for differentiation once in the appropriate media. The structure of these cells would differ during the process of differentiation accordingly.

Another ultrastructure feature of MSCs is the presence of vesicles in the cytoplasm. Intercellular communication can be mediated through direct cell–cell contact or transfer of secreted molecules. Recently, a third mechanism has emerged that involves intercellular transfer of extracellular vesicles. Cells release into the extracellular environment membrane vesicles either of endosomal origin or of plasma membrane origin. They are named exosomes and microvesicles, respectively [52]. In the study [38] carried out on isolated MSCs, showed vesicular trafficking. (Figure 10) These vesicles were prominent after the cells were cultured in a

media that stimulated its osteogenic differentiation. Microvesicles vary in size and are formed by the budding of the plasma membrane. Most cell types are known to produce microvesicles either constitutively or when stimulated during apoptosis or activation. The mechanisms involved in the mobilization of secretory microvesicles to the cell periphery, their docking, and fusion with the cell surface require the cytoskeleton (actin and microtubules), associated molecular motor proteins (kinesins and myosins) as well as other factors [53, 54]. The other clearly defined class of secreted membrane vesicles that originate from the endosomes are the exosomes. Exosomes were first discovered by Pan and Johnstone in 1983 [55]. They are formed by the invagination of endolysosomal vesicles to form multi-vesicular bodies. Exosomes are released by exocytosis. First, the cell membrane is internalized to produce endosomes. Subsequently, many small vesicles are formed inside the endosome by invaginating parts of the endosome membranes. Such endosomes are called MVBs. Finally, the MVBs fuse with the cell membrane and release the intraluminal endosomal vesicles into the extracellular space to

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Exosomes directly interact with the signaling receptors of target cells [57]. After that, the exosomes fuse with the plasma membrane of recipient cells and deliver their content into the cytoplasm [58]. Finally, the exosomes are internalized into the recipient cells. Once in the recipient cell, some of these engulfed exosomes may merge into endosomes and move across the recipient cells to be released into the neighboring cells. In the other case, endosomes fused from engulfed exosomes will mature into lysosomes and undergo degra-

Lipids and proteins are the main components. The protein content of exosomes from different cell types contains different endosome-associated proteins (e.g., RabGTPase, SNAREs, Annexins and flotillin). They are also enriched in proteins that associate with lipid rafts, including glycosylphosphatidylinositol-anchored proteins and flotillin [60]. The other main component of exosomes is the lipid. In comparison to the plasma membrane, exosomes are highly enriched in cholesterol, sphingomyelin and ceramides at the expense of phosphatidylcholine and phosphatidylethanolamine [52]. In addition to the proteins and lipids, various nucleic acids have recently been identified in the exosomal lumen, including mRNAs, microRNAs and other noncoding

The main functions of exosomes are their capacity to act as antigen-presenting vesicles, to stimulate immune responses [62]. Another main important feature of exosomes is being an ideal drug delivery vehicle. Meanwhile, research has been carried out encapsulating anticancer

The function of MSC-derived exosomes has not been well defined. They act as an intercellular communication vehicle for modulating cellular processes. It was recently revealed that exosomes derived from MSCs play important roles in mediating the biological functions of

A study demonstrated the electron microscopy of exosomes. They were cup-shaped and measured 40–100 nm in diameter. Exosomes are naturally secreted and well tolerated by the body. They are also safely stored and provide many therapeutic applications with avoiding the

become exosomes [56].

dation [57, 59].

RNAs [61].

MSCs [64].

drugs into exosomes [63].

Figure 8. Transmission electron micrograph of cultured human bone marrow derived stromal cells on day 21, showing large euchromatic nucleus (N) with clearly demarcated nucleolus (n). The cytoplasm shows mitochondria (M) [38]. Scale bar 1 μm.

Figure 9. Transmission electron micrograph of cultured human bone marrow derived stromal cells on day 21, showing part of its cytoplasm with multiple well-developed Golgi complexes (G) associated with large secretory vesicles (V), numerous mitochondria (M), and lysosomes (L). The cytoplasm shows profiles of rough endoplasmic reticulum (rER) [38]. Scale bar 0.5 μm.

Figure 10. Transmission electron micrograph of cultured human bone marrow derived stromal cells. The cytoplasm shows several cytoplasmic vesicles (Vs) of variable sizes. A coated pit (arrowhead) and numerous subplasmalemmal vesicles are also seen (thick arrows). A surface pseudopodium (Pd) is seen [38]. Scale bar 0.5 μm.

media that stimulated its osteogenic differentiation. Microvesicles vary in size and are formed by the budding of the plasma membrane. Most cell types are known to produce microvesicles either constitutively or when stimulated during apoptosis or activation. The mechanisms involved in the mobilization of secretory microvesicles to the cell periphery, their docking, and fusion with the cell surface require the cytoskeleton (actin and microtubules), associated molecular motor proteins (kinesins and myosins) as well as other factors [53, 54]. The other clearly defined class of secreted membrane vesicles that originate from the endosomes are the exosomes. Exosomes were first discovered by Pan and Johnstone in 1983 [55]. They are formed by the invagination of endolysosomal vesicles to form multi-vesicular bodies. Exosomes are released by exocytosis. First, the cell membrane is internalized to produce endosomes. Subsequently, many small vesicles are formed inside the endosome by invaginating parts of the endosome membranes. Such endosomes are called MVBs. Finally, the MVBs fuse with the cell membrane and release the intraluminal endosomal vesicles into the extracellular space to become exosomes [56].

Figure 8. Transmission electron micrograph of cultured human bone marrow derived stromal cells on day 21, showing large euchromatic nucleus (N) with clearly demarcated nucleolus (n). The cytoplasm shows mitochondria (M) [38]. Scale

Figure 9. Transmission electron micrograph of cultured human bone marrow derived stromal cells on day 21, showing part of its cytoplasm with multiple well-developed Golgi complexes (G) associated with large secretory vesicles (V), numerous mitochondria (M), and lysosomes (L). The cytoplasm shows profiles of rough endoplasmic reticulum (rER)

Figure 10. Transmission electron micrograph of cultured human bone marrow derived stromal cells. The cytoplasm shows several cytoplasmic vesicles (Vs) of variable sizes. A coated pit (arrowhead) and numerous subplasmalemmal

vesicles are also seen (thick arrows). A surface pseudopodium (Pd) is seen [38]. Scale bar 0.5 μm.

bar 1 μm.

36 Stromal Cells - Structure, Function, and Therapeutic Implications

[38]. Scale bar 0.5 μm.

Exosomes directly interact with the signaling receptors of target cells [57]. After that, the exosomes fuse with the plasma membrane of recipient cells and deliver their content into the cytoplasm [58]. Finally, the exosomes are internalized into the recipient cells. Once in the recipient cell, some of these engulfed exosomes may merge into endosomes and move across the recipient cells to be released into the neighboring cells. In the other case, endosomes fused from engulfed exosomes will mature into lysosomes and undergo degradation [57, 59].

Lipids and proteins are the main components. The protein content of exosomes from different cell types contains different endosome-associated proteins (e.g., RabGTPase, SNAREs, Annexins and flotillin). They are also enriched in proteins that associate with lipid rafts, including glycosylphosphatidylinositol-anchored proteins and flotillin [60]. The other main component of exosomes is the lipid. In comparison to the plasma membrane, exosomes are highly enriched in cholesterol, sphingomyelin and ceramides at the expense of phosphatidylcholine and phosphatidylethanolamine [52]. In addition to the proteins and lipids, various nucleic acids have recently been identified in the exosomal lumen, including mRNAs, microRNAs and other noncoding RNAs [61].

The main functions of exosomes are their capacity to act as antigen-presenting vesicles, to stimulate immune responses [62]. Another main important feature of exosomes is being an ideal drug delivery vehicle. Meanwhile, research has been carried out encapsulating anticancer drugs into exosomes [63].

The function of MSC-derived exosomes has not been well defined. They act as an intercellular communication vehicle for modulating cellular processes. It was recently revealed that exosomes derived from MSCs play important roles in mediating the biological functions of MSCs [64].

A study demonstrated the electron microscopy of exosomes. They were cup-shaped and measured 40–100 nm in diameter. Exosomes are naturally secreted and well tolerated by the body. They are also safely stored and provide many therapeutic applications with avoiding the

7. Conclusion

Abbreviations

ESCs embryonic stem cells

FSCs fetal stem cells

iPS induced pluripotent stem cells

MSCs mesenchymal/stromal cells

SEM scanning electron microscope

Rab Ras-related proteins in brain

GTP guanosine triphosphate

Amany A. Moneim Solaiman

University, Alexandria, Egypt

Medicine. 2008;2(4):169-183

TEM transmission electron microscope

SNARE soluble N-ethylmaleimide-sensitive factor receptor

Address all correspondence to<EMAIL_ADDRESS>
Department of Medical Histology and Cell Biology, Faculty of Medicine, Alexandria

[1] Bajada S, Mazakova I, Richardson JB, Ashammakhi N. Updates on stem cells and their applications in regenerative medicine. Journal of Tissue Engineering and Regenerative

CD cluster of differentiation

MMP metalloproteinases

P passage

Author details

References

promising candidate for drug delivery vehicle.

The MSCs maintained their undifferentiated histological structure till passage 9 for further tissue engineering. A detailed histological examination using the light and the electron microscopes is essential to understand the function of MSCs. In addition, exosomes represent a

Stromal Cell Ultrastructure

39

http://dx.doi.org/10.5772/intechopen.76870

Figure 11. Transmission electron micrograph of cultured human bone marrow derived stromal cells. The cell surface of shows an open fibripositors (short arrow) with large amounts of secretory product (S) is observed. Note the euchromatic nucleus (N) [38]. Scale bar 0.5 μm.

risk of immunological rejection and malignant transformation [65]. Therefore, the use of MSCs to produce exosomes for drug delivery is the subject of the day [66]. Recently, liposomes are preferred drug delivery systems. It is a synthetic vesicle with a phospholipid membrane that has the ability to self-assemble into various sizes and shapes in an aqueous environment [67].

Another morphological feature detected is pseudopodia-like structures extending from the cell membrane (Figures 7 and 10). This might explain the capacity of the cells for migration within the receiving tissue. The main role of these structures is to transmit the produced material from one cell into another by extending the pseudopodia and communicating cells with each other as well as in cell signaling [68]. Interestingly, one of the most striking features during differentiation is the observation of finger-like extensions of the plasma membrane known as fibripositors (Figure 11). These fibripositors were located at the side of the cell and protrude into the spaces between cells. These fibripositors are the site where collagen fibrils were located. It was reported that the initial stage of extracellular matrix deposition results in arrays of short collagen fibrils completely enclosed within these fibripositors. These fibrils are then subsequently deposited extracellularly [69, 70].

It was reported that fibrils leaving the fibripositors were seen to run along the external surface of the cell. Tracking of fibrils revealed that the collagen fibrils in fibripositors were shorter than those extracellularly. Thus, these data suggested that fibripositors might be a place of fibril assembly. They determined that short fibrils become longer inside closed fibripositors, then protruding fibripositors (open), often project into the matrix, releasing fibrils extracellularly where individual fibrils then coalesce into bundles. Thus, fibripositors are specialized sites not only of fibril assembly, but also share in fibril transport extracellularly [71].

Another study declared that the fibripositors are dynamic structures and their formation and stabilization depend on the actin cytoskeleton [72]. This might explain the existence of the cytoskeletal filaments in the differentiating cells [38]. Accordingly, these cytoskeletal structures might be actin filaments. It is possible that fibripositors have been involved in the alignment of extracellular collagen fibrils in a parallel arrangement [73].