**5.1 A specific and relevant topic**

Throughout the interviews, it is pointed out that successful citizen engagement processes are characterized by their being perceived as concrete and relevant to the participants. This means that the topic is not abstract but is based on the daily lives and interests of the citizens. This point is reflected in the observation of the consistent emphasis that residents of the area come to meetings largely because of specific matters that interest them. Thus, the focus of successful processes is directed towards themes that have a significant influence on the daily life of the residents. The question in this context is, of course, what is contained in the specific and relevant process. This question seems to be answered from another perspective. Citizens will greatly assess participation in the process based on whether they can acquire a real, visible, and noticeable benefit from it in their daily lives. The following topics are, by extension, highlighted as particularly appealing:

To conclude the interview process, a joint day was held in the Nordgård School hall. Citizens, associations, networks, and organizations in the area were invited. During the day, the following questions were examined: What important questions about the area will help us get closer to our hopes and dreams for the area, are we concerned? The above focus on "the close" topics are supported here by the questions raised by the participants in a future day as crucial for the future of Gellerupparken.


**5. Pathogenicity of amoebiasis**

*E. histolytica* causes intestinal and extraintestinal amoebiasis based on the site of infection. Though most infections do not harm the host (asymptomatic infections), establishment in the colonic mucosa via the Galactose/N-acetyl Galactosamine inhibitable lectin (Gal-lectin) is a pre-requisite for the disease (Chadee et al., 1987). Pathogenic forms of the parasite are known to secrete enzymes that facilitate their invasion into the mucosa and sub-mucosa causing deep-flask shaped ulcers (Figure 3), and in some cases entering the circulation and reaching internal organs like the liver, lungs, skin, etc. The disease in the colon is the most common with acute diarrhoea and dysentery accounting for 90% of the clinical amoebiasis cases (Espinosa-Cantellano and Martínez-Palomo, 2000) and only 1% involve the liver (Haque et al., 2003) (Figure 4).

#### **5.1 Asymptomatic colonization**

Asymptomatic infections are characterized by the parasite living in perfect harmony within the host. *E. histolytica* trophozoites have developed elusive tactics to prevent them from being purged from the host.. By modulating signals by intestinal epithelial cells (IEC), trophozoites direct anti-inflammatory host responses leading to a tolerogenic/hyporesponsive immune state favourable to their survival (Kammanadiminti and Chadee, 2006). Furthermore, products secreted by non-pathogenic *E. histolytica* strains normally disrupt and suppress NFκB signaling and as a result diminish pro-inflammatory responses normally detrimental to the parasite (Artis, 2008). Interleukin 10 (IL-10), an antiinflammatory cytokine, has been shown to play a significant role in maintaining this hyporesponsive state. On the other hand, a deficiency of IL-10 more often than not predisposes the host to develop the clinical amoebiasis (Hamano et al., 2006)

#### **5.2 Intestinal amoebiasis**

After an incubation period of 1-4 weeks, the parasite invade the colonic mucosa, producing characteristic ulcerative lesions and a profuse bloody diarrhea (amoebic dysentery). Amoebic invasion through the mucosa and into the submucosa is the hallmark of amoebic colitis. Contact of the trophozoites via the Gal/GalNAc lectin triggers a signaling cascade initiating the death of the host cell through different mechanisms such as phagocytosis, cytotoxicity and caspase activation instigating the invasive (intestinal and/or extraintestinal) stages of the disease. Other molecules involved in the disease process include: a serine-rich *E. histolytica* protein (SREHP), amoebapores, and cysteine proteases (Boettner at al., 2002; Mortimer and Chadee, 2010). Activation of damaging inflammatory and non-

adequate for the specific amplification of the desired genetic material. Considering these advantages, the LAMP assay could be a useful and valuable diagnostic tool particularly in developing countries where most of the infections are common as well as in hospital laboratories. Recently this method was developed specifically for the detection of *E. histolytica* (Liang et al., 2009). The efficiency of the developed method was compared to that of existing PCR methodology and was similar in terms of sensitivity and specificity. This method needs further evaluations to be used in local conditions in Africa in order to

*E. histolytica* causes intestinal and extraintestinal amoebiasis based on the site of infection. Though most infections do not harm the host (asymptomatic infections), establishment in the colonic mucosa via the Galactose/N-acetyl Galactosamine inhibitable lectin (Gal-lectin) is a pre-requisite for the disease (Chadee et al., 1987). Pathogenic forms of the parasite are known to secrete enzymes that facilitate their invasion into the mucosa and sub-mucosa causing deep-flask shaped ulcers (Figure 3), and in some cases entering the circulation and reaching internal organs like the liver, lungs, skin, etc. The disease in the colon is the most common with acute diarrhoea and dysentery accounting for 90% of the clinical amoebiasis cases (Espinosa-Cantellano and Martínez-Palomo, 2000) and only 1% involve the liver

Asymptomatic infections are characterized by the parasite living in perfect harmony within the host. *E. histolytica* trophozoites have developed elusive tactics to prevent them from being purged from the host.. By modulating signals by intestinal epithelial cells (IEC), trophozoites direct anti-inflammatory host responses leading to a tolerogenic/hyporesponsive immune state favourable to their survival (Kammanadiminti and Chadee, 2006). Furthermore, products secreted by non-pathogenic *E. histolytica* strains normally disrupt and suppress NFκB signaling and as a result diminish pro-inflammatory responses normally detrimental to the parasite (Artis, 2008). Interleukin 10 (IL-10), an antiinflammatory cytokine, has been shown to play a significant role in maintaining this hyporesponsive state. On the other hand, a deficiency of IL-10 more often than not

After an incubation period of 1-4 weeks, the parasite invade the colonic mucosa, producing characteristic ulcerative lesions and a profuse bloody diarrhea (amoebic dysentery). Amoebic invasion through the mucosa and into the submucosa is the hallmark of amoebic colitis. Contact of the trophozoites via the Gal/GalNAc lectin triggers a signaling cascade initiating the death of the host cell through different mechanisms such as phagocytosis, cytotoxicity and caspase activation instigating the invasive (intestinal and/or extraintestinal) stages of the disease. Other molecules involved in the disease process include: a serine-rich *E. histolytica* protein (SREHP), amoebapores, and cysteine proteases (Boettner at al., 2002; Mortimer and Chadee, 2010). Activation of damaging inflammatory and non-

predisposes the host to develop the clinical amoebiasis (Hamano et al., 2006)

improve the understanding of amebiasis in the continent as well as elsewhere.

**5. Pathogenicity of amoebiasis**

(Haque et al., 2003) (Figure 4).

**5.2 Intestinal amoebiasis**

**5.1 Asymptomatic colonization**

inflammatory responses following contact of the trophozoites to the gut wall induces a massive neutrophil infiltration across the epithelium into the underlying tissues resulting in weakening of epithelial cells and the mucous layer and allowing trophozoites to invade the intestinal epithelium and disseminating to other bodily sites (Ackers and Mirelman, 2006). The ulcers formed may be generalized involving the whole length of the large intestine or they may be localized in the ileo-caecal or sigmoido-rectal regions. Ulcers are normally disconnected with sizes varying from pin-head size to more than 2.5 cm in diameter. They may be deep or superficial. Base of the deep ulcers is generally formed by the muscularis layer. Nonetheless, superficial ulcers do not extend beyond the muscularis layer. A large number of fatalities results from perforated colons with concomitant peritonitis. *E. histolytica* also causes amoebomas. These are pseudotumoural lesions, whose formation is associated with necrosis, inflammation and oedema of the mucosa and submucosa of the colon. These granulomatous masses may obstruct the bowel.

Fig. 3. "Flask-shaped" ulcer of invasive intestinal amebiasis (hematoxylin-eosin, original magnification ×50). Source: **Pritt B S , Clark C G Mayo Clin Proc. 2008;83:1154-1160: Mayo Clinic Proceedings**

While the serine rich *E. histolytica* protein (SREHP) have been shown to promote adhesion of the trophozoites to host cells, cysteine proteases (CP), are known for their virulence in other protozoa as well as in tumour metastasis. Five *E. histolytica* proteins (EhCP1, 2, 3, 5 and 112) have been identified. All are alleged to play a role in the destruction of host cells, phagocytosis, together with the recruitment of neutrophils and macrophages and the induction of intestinal

Amoebiasis in the Tropics: Epidemiology and Pathogenesis 215

without direct contact (Stanley 2003). From the liver, *E. histolytica* trophozoites may enter

Fig. 4. Amoebic Liver abscess. Gross specimen of liver tissue with an abscess (white) that

**6. Role of genetic characteristics of the infecting strains in the pathogenesis**

The outcome of an infection may depend on several factors among which the genetic characteristics of the specific pathogen have been identified as an important one. Few polymorphic genetic loci have been identified and targeted to aid in the study of the population structure of *E. histolytica* strains and their possible relationships with the parasite's virulence and disease outcome (Clark, 2006; Paul et al., 2007). Examples of these genetic markers include protein coding genes (serine – rich *E. histolytica* protein, [SREHP] and Chitinase) and non-coding DNA (Strain Specific Gene and tRNA gene linked short tandem repeats [STR]) of PCR-amplified genes (Haghighi et al., 2003; Samie et al., 2008). In a study in Bangladesh, the tRNA-linked STR genotyping system has provided evidence that the parasite genome does influence the outcome of infection. tRNA-linked STR genotyping was also behind the recent observation of differences between parasite genotypes in the intestine and the liver abscess of same patients (Ali et al., 2007). Few studies, albeit inconclusive, using the polymorphic SREHP marker have indicated that certain SREHP profiles might be responsible for the presentation of intestinal amoebic symptoms (Ayehkumi et al., 2001; Samie et al., 2008). Yet, all studies with SREHP marker did support previous findings of extensive genetic diversity among *E. histolytica* isolates from the same

formed due to infection of the organ with *Entamoeba histolytica*. Source:

http://www.sciencephoto.com/media/250248/enlarge

**of amoebiasis**

into the general circulation and reach other organs (Figure 4).

inflammation (Mortimer and Chadee, 2010). Moreover, EhCP5 has also been shown to perform a variety of functions such as evasion of the host complement and immune system by preventing the activation of the classical complement system via the inactivation of IgG and the degradation of IgA (Laughlin and Temesvari, 2005).

Equally important in the pathogenesis and virulence of *E. histolytica* is the role of the phagosome-associated proteins. Many have been identified and their function in endocytosis and pathogenesis has been established. Examples include: EhRacA, EhRacG, EhPAK, actin and several Rab7-related GTPases (Laughlin and Temesvari, 2005). Cytokines such as IL-1β, IL-1α, IL-8 and TNF-α are suspected of aggravating the disease process and driving the immunopathogenesis mechanism (Kammanadiminti et al., 2003). Although neutrophils are known to cause intestinal tissue damage they are nevertheless critical for controlling the infection. Nonetheless, host and/or parasite factors normally play a role in determining whether the parasite is cleared or the disease becomes established (Asgharpour et al., 2005).

Although most intestinal invasions heal following an acute inflammatory response, *E. histolytica* evades destruction in a modest number of individuals and a chronic state is established. This chronic state is associated with the development of a non-protective adaptive immune response. Human data, in vitro and in vivo models support a paradigm that Th1 responses in the gut clear *E. histolytica*, while Th2 responses through the production of IL-4 are anti-protective, likely through suppressing IFN-γ. It is not yet clear what signals drive an anti-protective Th2 immune response instead of an effective protective Th1 response towards the infection. Evidence suggesting that genetics, the MHC restriction, nutrition and bacterial flora might play a role in directing the immune response towards *E. histolytica* infection exists. For example, the MHC class II allele DQBl\*0601 was reported to be associated with resistance to *E. histolytica* (Mortimer and Chadee, 2010). Susceptibility to ALA has been found to be associated with HLA-DR3 and complotype SC01 in some Mexican populations; this association is not seen for amoebic colitis or asymptomatic colonization with *E. histolytica* (Stanley, 2003).

#### **5.3 Extraintestinal amoebiasis**

About 5% individuals with intestinal amoebiasis, 1-3 months after the disappearance of the dysenteric attack, develop extraintestinal amoebiasis. Once in the blood, the parasite uses many different strategies to avoid elimination by the host and reaches other sites in the body (such as the liver, lungs, brain, etc). The most common extraintestinal site affected by the parasite is the liver and an Amoebic liver abscess (ALA) is its most common manifestation, predominantly seen in adult males. This chronic stage of ALA is characterized by defective cell-mediated immunity and the suppression of T cells and their defective proliferative responses (Campbell et al., 1999). *E. histolytica* trophozoites reaching the liver create their unique abscesses, which are well circumscribed regions of cytolysed liver cells, liquefied cells, and cellular debris. The lesions are surrounded by connective tissue enclosing few inflammatory cells and trophozoites. Parenchymal cells adjacent to the lesion are often unaffected. However, lysis of neutrophils by *E. histolytica* trophozoites might release mediators that lead to the death of liver cells, and extend damage to hepatocytes not in direct contact with the parasite. Studies have shown that in ALA in mice, most hepatocytes die from apoptosis, but necrosis is also present. In ALA from humans, the small numbers of amoebas relative to the size of the abscess suggests that *E. histolytica* can kill hepatocytes

inflammation (Mortimer and Chadee, 2010). Moreover, EhCP5 has also been shown to perform a variety of functions such as evasion of the host complement and immune system by preventing the activation of the classical complement system via the inactivation of IgG and

Equally important in the pathogenesis and virulence of *E. histolytica* is the role of the phagosome-associated proteins. Many have been identified and their function in endocytosis and pathogenesis has been established. Examples include: EhRacA, EhRacG, EhPAK, actin and several Rab7-related GTPases (Laughlin and Temesvari, 2005). Cytokines such as IL-1β, IL-1α, IL-8 and TNF-α are suspected of aggravating the disease process and driving the immunopathogenesis mechanism (Kammanadiminti et al., 2003). Although neutrophils are known to cause intestinal tissue damage they are nevertheless critical for controlling the infection. Nonetheless, host and/or parasite factors normally play a role in determining whether the parasite is cleared or the disease becomes established (Asgharpour

Although most intestinal invasions heal following an acute inflammatory response, *E. histolytica* evades destruction in a modest number of individuals and a chronic state is established. This chronic state is associated with the development of a non-protective adaptive immune response. Human data, in vitro and in vivo models support a paradigm that Th1 responses in the gut clear *E. histolytica*, while Th2 responses through the production of IL-4 are anti-protective, likely through suppressing IFN-γ. It is not yet clear what signals drive an anti-protective Th2 immune response instead of an effective protective Th1 response towards the infection. Evidence suggesting that genetics, the MHC restriction, nutrition and bacterial flora might play a role in directing the immune response towards *E. histolytica* infection exists. For example, the MHC class II allele DQBl\*0601 was reported to be associated with resistance to *E. histolytica* (Mortimer and Chadee, 2010). Susceptibility to ALA has been found to be associated with HLA-DR3 and complotype SC01 in some Mexican populations; this association is not seen for amoebic colitis or asymptomatic

About 5% individuals with intestinal amoebiasis, 1-3 months after the disappearance of the dysenteric attack, develop extraintestinal amoebiasis. Once in the blood, the parasite uses many different strategies to avoid elimination by the host and reaches other sites in the body (such as the liver, lungs, brain, etc). The most common extraintestinal site affected by the parasite is the liver and an Amoebic liver abscess (ALA) is its most common manifestation, predominantly seen in adult males. This chronic stage of ALA is characterized by defective cell-mediated immunity and the suppression of T cells and their defective proliferative responses (Campbell et al., 1999). *E. histolytica* trophozoites reaching the liver create their unique abscesses, which are well circumscribed regions of cytolysed liver cells, liquefied cells, and cellular debris. The lesions are surrounded by connective tissue enclosing few inflammatory cells and trophozoites. Parenchymal cells adjacent to the lesion are often unaffected. However, lysis of neutrophils by *E. histolytica* trophozoites might release mediators that lead to the death of liver cells, and extend damage to hepatocytes not in direct contact with the parasite. Studies have shown that in ALA in mice, most hepatocytes die from apoptosis, but necrosis is also present. In ALA from humans, the small numbers of amoebas relative to the size of the abscess suggests that *E. histolytica* can kill hepatocytes

the degradation of IgA (Laughlin and Temesvari, 2005).

colonization with *E. histolytica* (Stanley, 2003).

**5.3 Extraintestinal amoebiasis**

et al., 2005).

without direct contact (Stanley 2003). From the liver, *E. histolytica* trophozoites may enter into the general circulation and reach other organs (Figure 4).

Fig. 4. Amoebic Liver abscess. Gross specimen of liver tissue with an abscess (white) that formed due to infection of the organ with *Entamoeba histolytica*. Source: http://www.sciencephoto.com/media/250248/enlarge


**5. Conclusions**

This study proposes and applies a systematic data analytic methodology to analyse the experimental data obtained from tests of HPC samples with different admixtures. In contrast with other relevant studies aimed primarily at performing experiments (and providing rationale for the experiments based on materials, physics, or chemistry) or identifying the optimal HPC admixture(s) for grouting concrete materials to be used in the sea (e.g., for the base construction of offshore wind turbines), the purpose of this study is to perform a thorough investigation of the experimental variables related to the testing data.

To achieve this purpose, a methodological framework is proposed. In order to generate comprehensive and in-depth views of the data, numerous methods are utilised, including *P-Co-Co, Cos-Sim*, SLR, and heatmap or heat-based tabularised visualisation. This approach is significantly different from those of other experimental-based research or admixtureselection studies. Highlights of the research activities, results, and insights gained are presented below.

transformation was confirmed by another eight correlation matrices with variable *X* in Equation (2) being converted using transforms (2)–(9) described in Section 2.3.4.

The methods used in this study are common, and utilising them for this study was proven to be effective. Therefore, it should also be valid to utilise the methodological framework proposed in this research for similar purposes, e.g., data analysis for other concrete samples with different admixtures. From this perspective, a future story upon taking the proposed framework is perhaps clear from scratch. When a construction project is launched, one first sees if the HPC material(s) is to be used.

As can be expected, if so (using HPC) and if the planned admixtures of the HPC samples to be tested are analogous to the ones being tested in this study, efforts related to testing the materials can be reduced considerably by either policy. The first policy is to use the direct testing results for the samples in the established knowledge base (if 'no testing' is allowed legally). The second is to determine the testing item to be 'predicted' (as desired), look up the knowledge base to know what any other testing item can predict it (and also the information about whether the prediction will be effective and/or accurate), and decide whether to really but safely save the money, and time in most of the cases, for making tests by anticipating the results for the testing item (if some regulated testing items are mandatory).

However, if so (using HPC) and the planned HPC samples to be tested are different (e.g., due to a diversified purpose of use), or if not so (using the non-HPC material), theoretically, the first step is to make similar tests for these samples, followed by using the proposed framework to conduct the thorough data experiments (in order to identify the relationship and the mutual predictive power between each pair of sample parameters). Then as can be imagined, another knowledge base for the samples included by this purpose of use can be established. Although the outcomes and the unanticipated insights gained may vary in different cases or contexts, after this one-time job, what follows is a similar policy-making problem (see the former paragraph). Anyhow, all this may support efforts to save the time required for pre-project experiments, especially when the project is urgent, which is a crucial but inevitable fact in the current A/E/C industry.

It should be noted that the applications of this framework are not limited to cases in which the source datasets for each measure are tested using conventional methods (i.e., a parameter of HPC samples may have more than one testing method). For example, the framework could be applied when a part of the data is obtained using NMR (nuclear magnetic resonance) [48,49]. In many countries, NMR equipment is legal and commercialised, and the data for some variables can be obtained [50,51]. Other than this, except for HPC, the question of 'can similar data analysis using this framework be carried out for other construction materials?' is also worth of exploration.

Future research directions are shown in Figure 5, wherein the aim of this study which is clearly differentiated from other relevant studies.

**Figure 5.** The role of this research for HPC studies.

In the figure, the experimental-oriented studies are focused on the performance of the HPC, the inclusion of parameters for developing experiments, and statistics and variability in the data. A critical element that future research directions rely upon is the testing data sets produced by this research. Following this step, there are clear boundaries among the three research directions. The materials, physics and chemistry studies research direction involves a significant amount of opportunity for future studies. However, the knowledge base constructed for the HPC testing parameters as an outcome of the data analytics studies may benefit advance data studies in the future as well as subsequent selection decision modelling studies and current cementitious material portfolio determination studies. The selection decision modelling studies aim to rank the HPC samples with different admixtures and select the optimal candidate using the scientific decision models; therefore, the knowledge base may provide a precise numerical foundation for these models (and added value from the expert knowledge of the researchers).

**Author Contributions:** Conceptualization, methodology, software, visualization, Z.-Y.Z.; data curation, investigation, supervision, validation, W.-T.K.; funding acquisition, project administration, writing—original draft preparation, writing—review and editing, Z.-Y.Z. and W.-T.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Ministry of Science and Technology (MOST, Taiwan, ROC), grant number MOST 110-2410-H-992-020 and 110-2637-E-992-001. The APC was funded by MDPI discount voucher and MOST 111-2410-H-992-011. Note that the institution 'Ministry of Science and Technology (MOST)' has been renamed as National Science and Technology Council (NSTC) in August 2022 in this area. However, we still keep the original funding number for these projects were approved before the transition.

**Data Availability Statement:** N/A. All related data for the results of this study are detailed in the appendix sections.

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


*2.1. Search Strategy and Eligibility*

We used the online database Web of Science (WOS) to identify research papers regarding interventions to treat children and adolescents with obesity. The search was conducted in June 2019 and included articles published between January 1991 and December 2018. The WOS database was selected because it is suitable for our objectives, including (1) 254 various disciplines [36], (2) Higher impact journals [37]. Moreover, WOS allows us to download a large amount of data with important information for our analysis, such as the number of times the papers were downloaded and research areas which were assigned by WOS.

The WOS database search, using keywords, was divided into two steps. Specialists in the field of nutrition and adolescent health found keywords related to "obesity" and "children". In step 1, they were used to collect research articles and reviews from WOS. The database search process is shown in Table S1. These papers were then downloaded under the .txt format and imported into STATA (version 14.0, STATA Corp., TX, the USA). Using a STATA syntax, we searched specifically for papers that included the terms "trial\*", and "intervention\*" in the title or abstract (step 2).

Papers written in languages other than English were excluded. Additionally, papers other than original research articles or reviews (e.g., editorial, book chapter, letters to the editor, or conference proceedings), and papers where the authors were anonymous or unlisted were also excluded from the current study (Figure S1).


**3. Results**

Table 1 shows the prevalence of depression according to the demographic characteristics of the participants. The prevalence of depression was in the range of 2.7–7.2%. Educational level and income level were significantly associated with depression. The prevalence of depression decreased significantly with increasing income and educational level (*p* < 0.01).

**Table 1.** Prevalence of depression in postmenopausal women according to demographic characteristics.

Note: a *p* is determined by Mantel–Haenszel chi-squared test.

The mean EQ-5D scores of the participants are listed in Table 2. The EQ-5D score decreased with increasing age (*p* for trend < 0.001). In addition, the EQ-5D scores increased with increasing BMI (0.90, 0.89, 0.86, and 0.83 for obese, overweight, normal weight, and underweight, respectively). The EQ-5D scores also increased with increasing educational level (*p* for trend < 0.001) and income level (*p* < 0.001).

Table 3 shows the participants' age, BMI, and EQ-5D score according to depression category. The mean age, BMI, and EQ-5D score of participants with depression were 64.7, 24.2, and 0.88, respectively. The mean age and BMI were not significantly different between participants with and without depression, whereas the EQ-5D score was significantly lower in the participants with depression (0.77 and 0.89, respectively; *p* < 0.001).

**Table 2.** Mean EQ-5D scores of postmenopausal women according to demographic characteristics.

Note: a *p* is determined by linear trend test.

**Table 3.** Descriptive characteristics of the participants by depression category.

Note: Data are mean ± standard deviation. \* *p* < 0.0001 by *t*-test.

Table 4 shows the ORs for disability. The adjusted ORs for disability for each EuroQol category were significantly related to the prevalence of depression after adjusting for age (model 1) or for all other potential covariates (model 2) (*p* < 0.001). Compared to participants without depression, the adjusted ORs for mobility, self-care, usual activity, pain/discomfort, and anxiety/depression were 2.49 (95% CI, 1.79–3.47), 2.52 (95% CI, 1.68–3.78), 3.86 (95% CI, 2.78–5.36), 2.14 (95% CI, 1.57–2.92), and 5.52 (95% CI, 4.04–7.55), respectively, in those with depression (*p* < 0.001) (model 2).

**Table 4.** Adjusted odds ratios (OR, 95% CIs) for disability for the EuroQol categories in postmenopausal women.

**Table 4.** *Cont.*

Note: Model 1 was adjusted for age; model 2 was adjusted for age, body mass index (BMI), educational level, andincome.


**5.10 Cardiovascular activity**

*M. alba* mostly used in eastern countries to treat cardiovascular diseases. Chinese people also use *M. alba* for decreasing blood pressure which can lead to cardiovascular disorder. It also helps to reduce serum cholesterol, hypertension and also prevents artherosclerosis [47]. From the root bark of *M. alba* morusinol is extracted which prevents collagen and arachidonic acid which induces TXB2 formation in cultured platelets known as the main causative agent of congestive cardiac failure. Mulberries contain Reveratrol, which is a very important flavonoid is found to increase the formation of Nitric oxide (NO) that acts as a vasodilator. Richness of flavanoids and vitamin C makes mulberry also ideal for treating other cold, flu-like problems [14].


**6. Deactivation of the Wnt/beta-catenin pathway induces activation of PPAR gamma**

Inhibition of Wnt/beta-catenin pathway leads to an increase in transcription of PPAR gamma. Activation of the Wnt/beta-catenin signaling leads to osteogenesis, but not to adipogenesis. The canonical Wnt/beta-catenin-PPAR gamma system regulates the molecular switching of osteablastogenesis versus adipogenesis [6]. Wnt signaling maintains preadipocytes in an undifferentiated state through inhibition of both adipogenic transcription factors C/EBP alpha and PPAR gamma. Deactivation of Wnt/beta catenin pathway and activation of PPAR gamma are observed in ARVD [4, 31]. Taken together, these studies suggest that the canonical Wnt/ beta-catenin signaling downregulates PPAR gamma expression, inhibition of Wnt/betacatenin signaling upregulates PPAR gamma expression and PPAR gamma agonists inhibit the canonical Wnt/beta-catenin pathway.


**3. Apoptosis-Inducing Effect**

There is a wealth of information pertaining to apoptosis in anti-cancer research, and the

apoptosis-inducing properties of carotenoids may be an important approach to chemo-prevention and/or chemotherapy. Carotenoids from terrestrial plants, such as Ά-carotene and lycopene, have been extensively studied and implicated as cancer preventive agents. Carotenoids from marine sources have received much attention, as they are structurally different from those found in terrestrial sources. Recent studies have found that carotenoids from algae, including fucoxanthin and peridinin, induce apoptosis in cancer cells [6–9,20,21].

We investigated whether various carotenoids present in marine sources are potentially involved in cancer-preventing action in human leukemia HL-60 cells. We found that the 11 carotenoids investigated, siphonaxanthin most potently at inhibiting the viability of HL-60 cells [22]. Compared to fucoxanthin, siphonaxanthin, at a concentration of 20 ΐM, markedly reduced cell viability as early as 6 h after treatment. The cellular uptake of siphonaxanthin was 2-fold higher than fucoxanthin, demonstrating a positive correlation between cellular uptake and cell viability. The apoptotic activity of siphonaxanthin was characterized by increases in TUNEL-positive cells, and by increased chromatin condensation in the cells. This induction of apoptosis was accompanied by decreased expression of Bcl-2, and subsequent increase in the activation of caspase-3. These observations indicate that siphonaxanthin may be a more potent growth-inhibitor in cancer cells than fucoxanthin, possibly due to the differences in the structure of their different functional groups. The presence of hydroxyl group on the 19th carbon atom appeared to contribute to the strong apoptosis-inducing effect. This is further supported by the fact that siphonein (Figure 1C), an esterified form of siphonaxanthin that does not contain an additional hydroxyl group, had a reduced inhibitory effect on cell viability.

Interestingly, siphonaxanthin has been reported to up-regulate the expression of death receptor 5 (DR5) [22]. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) promotes apoptosis selectively in tumor cells without much effect on normal cells by binding to the transmembrane receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 [23]. Thus, any chemotherapeutic agent that activates TRAIL-induced apoptosis in cancer cells may be an attractive strategy in anti-cancer research. Yoshida *et al.* reported that halocynthiaxanthin and peridinin sensitize cancer cells to TRAIL-induced apoptosis by up-regulating the expression of DR5 [21], which indicates that some carotenoids can induce apoptosis through this pathway. In our study, siphonaxanthin potently up-regulated the expression of DR5, but fucoxanthin did not [22]. Siphonaxanthin could be potentially useful as a chemopreventive and/or chemotherapeutic agent.

## **4. Anti-Angiogenic Effect**

Angiogenesis is characterized by the growth and remodeling process of the primitive network of blood vessels into a complex network. In this process, endothelial cells secrete proteases, migrate through the extracellular matrix, proliferate, and differentiate [24]. Angiogenesis is involved in many physiological and pathological situations. In normal adults, most vasculature is quiescent, with only 0.01% of endothelial cells undergoing division [25]. However, angiogenesis is an essential process in the female reproductive cycle, along with the remodeling and regeneration of tissues [26]. Pathological angiogenesis is implicated in the pathogenesis of many diseases, including cancer, atherosclerosis, diabetic retinopathy, and rheumatoid arthritis [27]. The newly-formed blood vessels promote cancer growth by supplying nutrients and oxygen and by removing waste products. Metastasis also depends on angiogenesis, as tumor cells are shed from a primary tumor and grow in their target organs [28]. Angiogenesis is activated under other pathological conditions, such as ocular and inflammatory disorders [29]. Hence, prevention of angiogenesis under pathological conditions is a promising approach in the prevention of cancer and other pro-angiogenic diseases. It has been reported that some natural products, such as vitamin B6 [30], algal polysaccharides [31], and nasunin [32], suppress angiogenesis. Given this background, studies on natural bioactive molecules from marine algae have reported that fucoidans, polysaccharides from marine brown algae, and fucoxanthin and its deacetylated product, fucoxanthinol, exert anti-angiogenic properties [14,33].

To evaluate the anti-angiogenic effect of siphonaxanthin from green algae, we examined its

anti-angiogenic effect in cell culture model systems and by employing *ex vivo* approaches using human umbilical vein endothelial cells (HUVECs) and rat aortic ring [34,35], respectively. Siphonaxanthin significantly suppressed HUVEC proliferation at relatively lower concentration of 2.5 ΐM, while its effect on chemotaxis was not significant [36]. In addition, siphonaxanthin exhibited a strong inhibitory effect on HUVEC tube formation in an *in vitro* angiogenesis model. Siphonaxanthin suppressed the tube length at a concentration of 10 ΐM, while no tube formation was observed at 25 ΐM, suggesting that this could be due to the suppression of angiogenic mediators. The *ex vivo* angiogenesis assay using rat aortic ring indicated reduced microvessel outgrowth in a dose-dependent manner, and the reduction was significant at concentrations of more than 2.5 ΐM.

To elucidate the molecular mechanism underlying the anti-angiogenic activity of siphonaxanthin compared to fucoxanthin, we focused on the vascular specific proangiogenic factor, vascular endothelial growth factor (VEGF), and the other angiogenic factors, such as fibroblast growth factors (FGFs) [37,38]. It has been shown that both siphonaxanthin and fucoxanthin suppress the mRNA expression of fibroblast growth factor 2 (FGF-2) and its receptor (FGFR-1), as well as their *trans*-activation factor, EGR-1 [39]. These suppressive effects of siphonaxanthin were more effective than fucoxanthin. However, the mRNA expression of VEGFR-2, a potent signal transducer involved in the

VEGF-mediated signaling pathway, was not significantly affected by these carotenoids. Furthermore, these two marine algal carotenoids can down-regulate the phosphorylation of FGF-2-mediated intracellular signaling proteins such as ERK1/2 and Akt. Inhibition of FGF-2-mediated intracellular signaling proteins by these carotenoids represses the migration of endothelial cells, as well as their differentiation into tube-like structures on Matrigel. These results demonstrate, for the first time, the possible molecular mechanism underlying the anti-angiogenic effect of these two algal carotenoids and suggest that their anti-angiogenic effect is due to the down-regulation of signal transduction by

FGFR-1 in vascular endothelial cells. Siphonaxanthin exhibited its anti-angiogenic effect at lower concentrations than fucoxanthin, indicating its potential as a strong angiogenesis inhibitor.

These findings provide new insights into the novel bio-functional property of marine algal carotenoids, especially that of siphonaxanthin, which has the potential to enhance current anti-angiogenic therapies in the treatment of cancer and other pro-angiogenic diseases.


**7. Cyanobacteria**

Cyanobacteria, known as blue-green algae, are ancient photosynthetic prokaryotes which inhabit a wide diversity of habitats including tropical reefs, fresh water ponds, streams and puddles and fresh water ponds. Luxuriant growth of cyanobacteria in these adverse environments conditions is based on their abilities of forming resistant spores, opportunistically colonizing micro-habitants and surviving under conditions of high UV-flux through production of UV-absorbing pigments, has made them one of the successful life forms on earth. Cyanobacteria associated with ascidians, their symbotic relationship was first found out in 1982 by Kott.

Cyanobacteria are phylum of bacteria that produce oxygen during photosynthesis. Host ascidians that exhibit symbiotic relationship with cyanobacteria, Prochloron, which belong to the Didemidae family and are therefore called as "Didemnin ascidians". Cyanobacterial symbionts can both provide nutrients by means of Carbon fixation, nitrogen recycling and metabolite production and also exhibits defensive reaction for the host ascidian. Ascidian host are capable of producing some of the nitrogen containing nutrients that are needed for the cyanobacterial symbionts growth and also protection against the ultra violet radiation. Additional feature is that, a rich source of biologically active products, has assisted some of these organism to survive in predator-rich tropical reef ecosystems. Tropical marine Cyanobacteria particularly the filamentous forms such as *Lyngbya* species or *Symploca* species have been a source of novel natural products with therapeutic and biotechnological potential. Marine cyanobacteria are considered to be an important source of structurally diverse and biologically active natural products. Different peptides isolated from a wide variety of marine cyanobacteria, induces

#### *Antimicrobial Peptides Derived from Ascidians and Associated Cyanobacteria DOI: http://dx.doi.org/10.5772/intechopen.99183*

anticancer effects on various human cell lines. Most studied cytotoxic cyanobacteria on human tumor cell lines inducing minimal inhibitory effects [53].

Cyanobateria have a rich complement of photosynthetic pigments, including chlorophyll a and b, as well as several accessory pigments (phycoerythrin, phycocyanin, and allophycocyanin). Phycoerythrin has found application in biotechnology as a conjugate to antibiotics that then allow visualization of cellular constituents and processes and chlorophyll is being explored for its cancer chemotherapeutic activity. Apratoxin A is a cyclic depsipeptide extracted from *Lyngbya majuscule*. Exhibited cytotoxic effects on Human HeLa cervical carcinoma cells by cell cycle inhibition [54]. Similar mechanism was also reported on cyclic depsipeptide Coibamide A, isolated from *Leptolyngbya* species on Human lung cancer cell line and Lyngbyabellin B isolated from *Lyngbya majuscule* on Human Burkitt [55] lymphoma cells. Dolastatin 10 and Symplostatin 1, isolated from *Symploca* species, showed cytotoxic effect on human lung cancer cell line and Human breast carcinoma cell line by both Bcl-2 phosphorylation and Caspase-3 protein activation. Anticancer peptides such as *Lyngbya sp* and *Nostoc sp*, shows activity against cancer on different cell lines through microfilament disruption, secretory pathway inhibition.

Cyanobacteria inhibit Gram-negative and Gram-positive pathogenic bacterial species. Extracts of *Cylindrospermopsis raciborskii*, CYP011K and *Nostoc* species,

CENA69 possibly caused cancer cell inhibition. Extracts from *Fischerella* species, CENA213 showed inhibition of 3LL lung cancer cells. NPLJ-4 extracts isolated from *M.aeruginosa* reported to have inhibition against CT26 colon cancer cells. All of these extracts are prone to have low inhibitory activity towards human peripheral blood lymphocytes.

*Aphanazomenon flos-aquae*, freshwater cyanobacteria reported to contain immune stimulating properties, Other cyanobacteria Spirulina, a rich source of digestable proteins with a complete complement of essential amino acids [56].

*Lyngbya majuscule* from Curacao, yielded metabolites with broad biological properties, including those with toxicity to arthropods, those toxic to fish and those toxic to gastropods.

Bisanthrantaquinones, isolated from blue green algae associated with the colonial ascidian *Ecteinascidia turbinate*. These are the antimicrobial metabolites from ascidian-associated cyanobacteria (**Figure 7**), available to date. It has greater antibacterial effectiveness, and has resistance towards multi-drug resistant bacteria and vancomycin-resistant *Enterococcus faecalis* with an MIC of 0.6 and 12uM [57].

Cyanobacteria are considered to be an important source of bioactive metabolites, with various aspects of cytotoxic, antiviral, anticancer, antimitotic, antimicrobial, specific enzyme inhibitor and immunosuppressive activity. Cyanobacteria holds the presence of non-ribosomal peptide synthetase and polyketide synthetase genes, owing it to be the potential for finding novel natural drug products from these organism. Thus, cyanobacterium species are a rich strain enriched with the source of natural products with potential for pharmacological and biotechnological applications.

#### **7.1 Tubulin binding proteins**

Microtubules play many significant roles in cell biology. Formation of microtubules results from the polymerization of the subunit protein tubulin, first into heterodimers subsequently binds end to end with other heterodimer forming a protofilament, which in turn interacts to form sheets and eventually microtubules. Specifically, assembly and motility are crucially for the formation of the spindle apparatus during cell replication and mitosis where microtubule fibers direct the separation of sister chromatids into the resulting daughter cells. In case of rapidly

**Figure 7.** *Image showing marine cyanobacteria.*

dividing cancer cells, microtubule assembly has been an important target in the development of new chemotherapeutic agents. Various drugs have developed to disrupt the process of mitosis and cause catastrophic cell death by either stabilizing microtubule complexes, caused by taxol. Depolymerization of the tubulin protein complex caused by Vinblastin [58]. Recently antimicrobial peptides targeting intracellular tubulin has developed from marine natural products.

Pharmacological properties of the marine mollusk, derivatives of *Aplysiidae* commonly know as the 'sea hare' or 'nudibrunch' has been reported for their toxic secretions. Biological activity of the cyanobacteria present in sea hare *Dolabella auricularia*, its structure consists of an active constituent, dolastatin 10. Dolastatin 10 displayed exceptional activity against the P388 lymphocytic leukemia cell line with ED50 value of about 4.6\*10-5 g/ml. It is also reported to have potent antineoplastic activity. Dolastatin 10 isolated from field collections of the marine cyanobacterim *Symploca* species, clarifying the concept that the true biosynthetic source is the cyanobacteria and not the sea hare. Dolastatin 10, a unique linear pentapeptide is composed of four novel amino acid residues such as dolavaline, dolaisoleucine, dolaproline, dolaphenine and valine. High resolution mass spectroscopic analysis such as 1H (COSY, 2D-J resolved) and 13C NMR methodologies revealed the structure of this linear pentapeptide. This peptide sequence was assigned on the basis of several low resolution mass spectral fragmentation techniques. This peptide was found to be involved in the binding of Dolastatin 10, whereas other antimicrobial peptides near the exchangeable nucleotide and vinca alkaloid sites on microtubules [59]. Dolastatin 10 inhibits microtubule assembly in vitro and subsequently blocks cytokinesis. Also this peptide was noted for its non-competitive inhibition of radiolabeled vinblastine and vincristine to tubulin as well as tubulin dependent hydrolysis of GTP.

Dolastatin 10 binds to the 'peptide groove' lying within the r- subunit of tubulin. Molecular modeling suggested that the chiral centers of dolavaline, valine and dolaisoleucine binds in a manner that require the dolaphenine moiety to sterically block access to the vinca alkaloid and exchangeable nucleotide binding sites. Evidence of noncompetitive inhibition of Vinca alkaloid binding was realized by observation that tubulin polymerization and nucleotide binding are substantially diminished at sub-stoichiometric concentrations of dolastatin 10.

Dolastatin 10, on its Phase II clinical trial proved to be an antitumor agent, by evaluating its antitumor efficacy in patients with measurable recurrences of platinum –sensitive ovarian carcinoma in relation to the degree of toxicity [60]. *Antimicrobial Peptides Derived from Ascidians and Associated Cyanobacteria DOI: http://dx.doi.org/10.5772/intechopen.99183*


Nanometric 3D Printing of Functional Materials by Atomic Layer Deposition

*David Muñoz-Rojas, Matthieu Weber, Christophe Vallée, Chiara Crivello, Abderrahime Sekkat, Fidel Toldra-Reig and Mikhael Bechelany*

## **Abstract**

Atomic layer deposition (ALD) is a chemical vapour deposition (CVD) method that allows the layer-by-layer growth of functional materials by exposing a surface to different precursors in an alternative fashion. Thus, thanks to gas-solid reactions that are substrate-limited and self-terminating, precise control over thickness below the nanometer level can be achieved. While ALD was originally developed to deposit uniform coatings over large areas and on high-aspect-ratio features, in recent years the possibility to perform ALD in a selective fashion has gained much attention, in what is known as area-selective deposition (ASD). ASD is indeed a novel 3D printing approach allowing the deposition of functional materials (for example metals to oxides, nitrides or sulfides) with nanometric resolution in Z. The chapter will present an introduction to ALD, which will be followed by the description of the different approaches currently being developed for the ASD of functional materials (including initial approaches such as surface pre-patterning or activation, and newer concepts based on spatial CVD/ALD). The chapter will also include a brief overview of recent works involving the use of ALD to tune the properties of 3D printed parts.

**Keywords:** spatial atomic layer deposition, spatial chemical vapor deposition, area-selective deposition, atomic layer infiltration, surface nanoengineering, 3D printing of functional materials

## **1. Introduction**

Vapor-phase techniques are powerful approaches for the deposition of functional thin films of different materials, including metals and compounds such as oxides, nitrides, and even organic materials and composites onto a substrate [1–3]. There are two types of vapor deposition methods, namely physical and chemical. Physical vapor deposition (PVD) methods involve a change of state (i.e., evaporation and recondensation) of a source, and include, among other, sputtering, pulsed laser deposition or different evaporation approaches [4]. While PVD methods yield materials of high quality with tunable properties, they are performed in high vacuum and often high temperatures, using sophisticated equipment. Finally, the

low vacuum process results in a line-of-sight type of coating (i.e., only taking place on the directly exposed surfaces.

The possibility to pattern and 3D print materials at different scales has a tremendous impact on many technologies and applications. Over the years. Different 3D printing approaches have been developed allowing such patterning. This include, to name a few, aerosol jet printing (also known as Maskless Mesoscale Materials Deposition or M3D) [5], ink jet and screen printing [6], laser chemical vapor deposition (LCVD) [7], laser-induced forward transfer (LIFT) [8, 9] or micro stereo lithography and multiphoton lithography [10]. The interested readers are encouraged to the cited references for more details on these methods. In this chapter, we focus on 3D printing approaches based on ALD. A brief introduction to CVD and ALD is thus presented next.

Chemical vapor deposition (CVD) approaches on the other hand rely on chemical reactions between different precursors on and over a surface. In conventional CVD, the precursors are injected in the reactor at the same time and the reaction is activated by heat (hot substrate) or by other energy sources, such as plasma. A scheme representing the reaction chamber is shown in **Figure 1a** [11]. This technique allows the deposition of high-quality films [12], and is largely used by the industry. Nonetheless, CVD is governed by the diffusion of the different gas precursors, and therefore, the deposition of extremely thin films with a thickness control at the sub-nanometer level [13], and the uniform coating of large areas or high-aspect-ratio/porous features is extremely difficult [14].

Such limitations prompted the development of an alternative method, namely, atomic layer deposition (ALD). ALD is indeed a CVD method but it is characterized by having the substrate exposed to the different precursors one at a time, and not simultaneously as in CVD. Thus, in typical ALD processes, a precursor is first injected in a deposition chamber where the substrate is located. The precursor can then react with active sites on the surface (i.e., undergoing a chemisorption) until the latter is saturated. A purge step is then applied to eliminate excess precursor and reaction byproducts. Then a second reactant is injected that will react with

#### **Figure 1.**

*(a) CVD mechanism where the precursor is adsorbed on the surface at relatively high-temperature followed by the film growth and a release of volatile byproducts, (b) ALD process: Schematic of one ALD cycle of monolayer growth. The first step consists in exposing the substrate to the precursor followed by a purge step to remove all the byproducts an excess precursor, then another step with a co-reactant agent and the final step in which the byproducts an excess precursors are purged again, (c) illustration of edge coverage for ALD, CVD, and PVD.*

#### *Nanometric 3D Printing of Functional Materials by Atomic Layer Deposition DOI: http://dx.doi.org/10.5772/intechopen.101859*

the preciously adsorbed layer. After the reaction is completed, again a purge step is necessary to eliminate excess reactant and reaction byproducts. Such an ALD cycle is shown in **Figure 1b**. As a result of this sequential exposure to the different reactants, the ALD process is surface-selective and self-terminating, which in turn offers unique control over film thickness at the angstrom level (i.e., a given growth per cycle, GPC, being obtained for each process as a function of the reactor geometry and precursors used) and allows the conformal coating of porous, complex and high-aspect-ratio substrates. The films are also compact and free of pinholes and can be obtained at low temperatures (even room temperature) due to the high reactivity of ALD precursors. The reader is referred to reviews and books dedicated to ALD for more information [1–3]. **Figure 1c** shows a sketch of the different types of coating obtained over the high-aspect-ratio features when using the different techniques discussed.

Over the years, the number of materials that can be deposited by ALD has grown enormously, including pure elements (e.g., metals), nitrides, sulfides, oxides, fluorides, etc. (see the atomic limits site, with includes an ALD materials database that is permanently being updated [15]). While at the origin the main motors of the ALD development were the deposition of homogeneous coatings over large areas or high aspect-ratio features, in the last years, there have been innovative developments in the ALD field that allow the localized and topological deposition of functional materials. This opens the door to its application as a new nano-to-macro 3D printing technology based on gas precursors. These recent developments, namely, area-selective deposition (ASD) and different spatial approaches, are presented in Sections 2 and 3, respectively. Finally, the unique assets of the ALD technique are ideal to tune the properties of pieces fabricated by conventional 3D printing approaches. Section 4 presents a brief overview of recent results on this line. The chapter finishes with some conclusive remarks.

## **2. Area-selective deposition (ASD)**

For more than 50 years, the shrinking of microelectronic devices has involved successive steps of deposition, lithography and etching. Indeed, unlike building a house, it is not possible to directly draw the walls or pillars of a chip on a 300 mm substrate. It is therefore necessary to first cover the whole substrate with a thin layer, before removing part of it by the steps of lithography (to draw the object) and etching (to remove what should not remain on the surface). This is called a topdown approach. The reduction of the dimensions of microelectronic devices in the last 10 years to nanometric scales has greatly complicated these steps and increased their cost. Indeed, for many years, the wavelengths used to draw were greater than the desired line thickness. It was therefore necessary to make lithography more complex by integrating etching/deposition steps to achieve the desired dimensions, such as multi-patterning (Self Aligned Double or Quadrupole Patterning—SADP and SAQP). Thus, these steps allowing to obtain locally nanometric materials on the substrate are now complex, time-consuming and expensive. They must also be done with nanometric placement precision, which is already a real challenge.

The alternative solution to this increasingly complex approach is to deposit the material directly and selectively on the desired surface without having to resort to lithography steps. This so-called selective growth on a surface is a bottom-up approach and is known as area-selective deposition (ASD) [16, 17]. In an ideal ASD process, a thin film should be uniformly deposited in the desired growth region while no deposition should be observed in the desired no-growth region. This requires the use of a surface selective deposition process, with controlled growth

at the atomic scale, and thus ALD is the one that seems to be the most adapted. Indeed, a growing number of researchers working on the ALD process are now trying to establish strategies from this process to have a material deposited selectively on a surface. The three main strategies are: (i) to use an inherent selectivity of the precursor/substrate couple [18, 19]; (ii) to block the growth on the no-growth area by a pre-deposition treatment [20, 21]; (iii) to promote the growth on the growth area by a pre-deposition treatment [22]. Whatever the strategy, we observe growth on all surfaces after a certain number of cycles, or at best, a little defectivity with nuclei on the no-growth area, i.e., the selectivity fades out during the successive ALD cycles. It was then proposed to regularly add the surface treatment step (passivation step) in the ALD cycles changing a cycle from a (treatment + AB) process to an (ABC) cycle with the treatment reinjected regularly [23]. Another proposed solution is to use super-cycles with the injection of etching steps every *x* ALD cycles, this is called ASD by super-cycles of deposition-etching [24–28]. In the end, with these different ASD strategies, passivation or super-cycles, [29] the possibility to deposit thin films of more than 10 nm on a previously selected surface has been successfully demonstrated. However, the additional steps (passivation, etching) increase the processing time. It takes more than 24 h for passivation by a selfassembled monolayer (SAM) using chemical baths. The insertion of etching steps in an ALD process also increases the processing time by a factor of 2–3 compared to a conventional ALD process. Finally, the insertion of passivation or etching chemistries can induce contamination of the deposited layer as well as process drifts (with for example a modification of the chemistry of the ALD reactor walls during the etching step). Nevertheless, ASD has the potential to deposit functional

#### **Figure 2.**

*(a) Illustration of four different strategies for an area selective deposition where a is the growth area and B the no-growth area; (b–d) examples of ASD using deposition and etch: (b) TiO2 on TiN vs. Si/SiO2 (reprinted with permission) [25], (c) patterning at the microscale of TiO2 and W films (reprinted with permission) [30], and (d) topographical selective deposition of Ta2O5 on Si (reprinted with permission) [31].*

*Nanometric 3D Printing of Functional Materials by Atomic Layer Deposition DOI: http://dx.doi.org/10.5772/intechopen.101859*

materials with complex 3D shapes and nanometric resolution, well beyond the possibilities offered by standard 3D printing methods. This can be done by different approaches, as shown in **Figure 2**.

Although, ASD offers a huge potential for the 3D printing of functional materials at resolutions orders of magnitude below what can be achieved with conventional 3D printing approaches, the different steps it implies (i.e., surface prepattering, regeneration of the selectivity) make them harder to work with. It would thus be desirable to develop an ALD approach that could allow the direct deposition of patterned materials. This can indeed be achieved, as detailed in the next section, by using different *spatial* approaches. The question that arises now is whether this approach can be used to develop ASD processes on larger surfaces and at dimensions that are no longer nanometric but micrometric or millimetric. The natural answer is yes. Indeed, an optimized ALD process is uniform across m-scale objects. Using a spatial ALD technology, we can then imagine selectively printing on a large surface with ALD: 3D printing. This is described in the next section.

## **3. 3D printing of functional materials based on spatial CVD/ALD approaches**

As explained in the introduction, the unique assets of ALD are the result of having a surface-limited, self-terminated reaction between gas reactants and the surface of a substrate. To limit the reaction to the surface, the ALD is based in alternate exposures of the precursors to the substrate. Traditionally, this has been done by sequential injection of the precursors in a deposition chamber followed by purging steps, thus in a temporal approach, as detailed in **Figure 1b** above and in the scheme below (**Figure 3a**). An alternative approach consists in having a continuous injection of the different reactants but in different locations of the reactor, keeping them separated by a region of inert gas. Then, by alternatively exposing the substrate to the different regions, the ALD cycle is reproduced (**Figure 3b**). This approach is known as Spatial ALD (SALD) [33–36]. The first advantage of processing in the spatial mode is that the process can become much faster (up to two orders of magnitude) since no purging step is required.

The SALD concept is very versatile and can indeed be applied in different ways [33, 37]. SALD can even be performed at atmospheric pressure (i.e., no vacuum processing) and even in the open air (i.e., no deposition chamber), and this is sometimes referred to as Atmospheric-Pressure SALD (AP-SALD). This is the case of the close-proximity approach based on a manifold injection head, originally presented by Kodak [38]. In this particular approach, the different reactants are carried to the injection head where they are distributed along alternate parallel channels (**Figure 3c**) [39]. By proper design of the head, the different flows can be kept separated provided the substrate is at close proximity of the head (i.e., 50–200 μm). Then by scanning the substrate back and forth under the head the ALD cycles are achieved. It is worth noting that since the size and area of the deposition depend on the head size and substrate scan distance, this SALD approach can already be seen as an ASD approach at the cm scale.

Close-proximity SALD approaches based on injection heads have several extra appealing advantages. The first one is that deposition can be also performed in spatial CVD (SCVD) mode. Then, crosstalk between the different reactants above the surface of the substrate is allowed. In this case, the deposition rate can be faster, but care must be taken since the properties of the materials deposited could change [32]. The impact of the change in the film properties when passing from the SALD to SCVD mode has to be evaluated depending on the intended application, but

#### **Figure 3.**

*(a) Schematic representation of the classical temporal ALD approach with the different characteristic steps of an ALD cycle: (1) injection of the first precursor, (2) purging step, (3) injection of the second precursor, (4) purging step, separated in time. (b) Schematic representation of the spatial ALD approach, where the precursors are injected continuously in the reaction chamber in different locations separated by an inert gas and the sample is exposed to the different regions to reproduce the ALD cycle. (c) Scheme of the close-proximity AP-SALD approach based on a manifold injection head: the precursors are carried out from the containers of the head where they are distributed in parallel alternative channels. (d) COMSOL simulation of the mass fraction of each precursor present in different areas of the substrate (left). In these cases the evacuation of the precursors is not efficient and thus cross-talk is observed, yielding a CVD reaction on the zones where the precursors meet (see COMSOL simulation in the center). If a deposition is made in static mode (i.e., without moving the substrate), 4 lines of oxide can be obtained, as shown in the optical image (right) where 4 lines of ZnO have been deposited on a Si wafer in this way (adapted from Ref. [32]).*

several works have demonstrated that the SCVD can be used to deposit components for functional devices [40]. In addition, the possibility of having SCVD opens the door to a new ASD approach. Indeed, the CVD reaction can be located in different areas above the substrate. **Figure 3d** presents a computational fluid dynamics (CFD) simulation that shows the areas over the substrate where the different reactants meet and thus react when the deposition is performed in certain SCVD conditions. Then, by performing a static deposition (i.e., without the substrate scan that is needed to perform the spatial ALD cycles) growth of the films can be localized to the regions where the reactants meet (see the four ZnO lines obtained by this approach in **Figure 3d**). This constitutes a new alternative approach of ASD at a higher scale and much faster deposition rate than the traditional ASD approaches based on ALD that have been described in the previous section [32].

The second advantage of using a close-proximity SALD approach based on an injection head is that the system can be customized by simply modifying the injection head. While this is so, the modification and fabrication of the head can result very difficult, if not impossible, thus limiting the potential of the approach (see **Figure 4a** where the scheme of a standard SALD head is shown. It comprises several parts that need to be fabricated separately and then soldered, and the distribution of the different gas flows to the head is quite complex involving many pipes). To overcome this limitation, D. Muñoz-Rojas' group at the Laboratoire des Matériaux et du Génie Physique (LMGP, Grenoble, France) has introduced the utilization of 3D printing for the fabrication of customized SALD injection heads [41]. This allows having more freedom to design the head and, for example, the gas distribution can be incorporated in the body of the head (**Figure 4b** and **c**) [41–43]. Plastic heads can be printed for depositions taking place at low temperatures while metal 3D printing is also possible for higher temperatures [44]. Thanks to 3D printing, the design of the heads can be easily customized. This is very convenient to easily

*Nanometric 3D Printing of Functional Materials by Atomic Layer Deposition DOI: http://dx.doi.org/10.5772/intechopen.101859*

#### **Figure 4.**

*(a) Scheme of a close-proximity SALD head made of several parts and fabricated by conventional approaches. (b) 3D scheme of a head design integrating the gas distribution for the different gases inside its body: metallic precursor in green, co-reactant in red, inert gas in blue and exhaust in black. (c) Head printed with clear resin where the distribution channels can be observed. (d) 3D scheme of a head designed for circular shape deposition in static SCVD mode. ZnO circles with different thicknesses are shown. (e) Picture of a printed SALD pen (left), bottom view of the concentric gas outlets in the SALD pen approach allowing deposition in any direction (right). (f) Scheme of the SALD pen installed in a 3D table. (g) Scheme of a SALD pen implemented in the XYZ table and drawing ZnO in a circular pattern. (h) LMGP initials on a Si wafer drawn with the 3D printed SALD pen (adapted with permission from Ref. [41]).*

modify the area of deposition, and also to have free-form patterns when performing SCVD with custom heads (**Figure 4d**) [41].

The possibility to deposit free-form patterns without having to modify the head for each design would also be appealing. This can indeed be done if instead of using parallel channels, the head is designed so that concentric channels are used. In this way, no matter which direction the head moves, the substrate will be exposed to the different reactants, thus leading to ALD film growth (**Figure 4e**).  D. Muñoz-Rojas' group demonstrated that such a SALD pen can be printed and used to deposit free-form patterns when installed in an XYZ table, in this case with a resolution going down to several mm (**Figure 4f-h**) [41]. This represents a new 3D printing approach that is based on gas precursors and that offers nanometric resolution in Z. Here again, the resolution of the obtained patterns in X-Y depends on the head design and the possibility to scale it down. Indeed, the latter work by Midani et al. presented a similar concept in which sub-millimiter resolution was achieved by inserting a capillary in the central metal precursor channel of the SALD pen [45].

#### *Advanced Additive Manufacturing*

Certainly, the advances in the different 3D printing technologies will allow de fabrication of SALD heads with smaller channels, which will extend the possibilities of SALD for depositing patterns of functional materials down to the micrometer scale in X-Y.

## **4. ALD for conventional additive manufacturing: recent works involving the use of ALD to tune the surface and properties of 3D printed parts**

Additive manufacturing (AM), also known as 3D printing, is recognized as a revolutionary technology, which has primarily been used in the field of engineering to create customized prototypes [46–48]. 3D printing has now become a subject of great interest and is extensively applied in many areas, such as prototyping, medicine [49] or aerospace [50], since it allows new products with complex geometries and microarchitecture (multiple pore shape and size) to be imagined, designed and fabricated. However, the material from which the designed products are made is still limited by the 3D-printing material itself. Even if the number of available materials that can be printed is expanding [48], most of the manufactured objects are made of polymer or stainless steel. Thus, a post-treatment may be required to control the nature and chemistry of the product surface and offer it its desired functionality. As illustrated in **Figure 5** and discussed below, ALD is a highly appealing technique to expand the potential of 3D printing through coating or infiltration of the printed parts.

As seen previously, ALD can be used as an innovative and novel 3D printing route, to prepare customized and complex 3D structures at the nano-to-cm scale. In addition, this technology can also be used to precisely tune the surfaces of 3D printed objects that were manufactured using more "conventional" additive manufacturing approaches such as fused deposition modeling, inkjet printing, stereolithography, selective laser sintering (SLS), powder bed fusion or even bioprinting [46–48]. ALD allows the preparation of thin films with a sub-nanometer thickness control, high uniformity and excellent conformality even on high aspect ratios substrates, a unique capability, as discussed in the first section of this chapter [3, 53–58]. As ALD allows the conformal coating of complex substrates with nanolayers made of an expanding

#### **Figure 5.**

*Illustration of the combination of 3D printing and atomic layer processing. Depending on the 3D printed material, either a coating is obtained, allowing for the tuning of the surface properties (typical ALD); or inorganic components are introduced to the subsurface of the 3D objects (atomic layer infiltration, ALI [51, 52]), permitting the tuning of the matrix.*

#### *Nanometric 3D Printing of Functional Materials by Atomic Layer Deposition DOI: http://dx.doi.org/10.5772/intechopen.101859*

number of materials [2, 15], such as oxides [59, 60], metals [61], nitrides [62] and sulfides [63], the combination of this route with 3D printing can be beneficial to a myriad of applications.

A large number of 3D printed objects are made of polymers, the current mainstream materials being ABS (acrylonitrile butadiene styrene) and PLA (polylactic acid). However, when performing ALD on 3D printed objects based on such polymeric materials, some considerations must be taken. The first obvious consideration is related to the ALD process temperature, which has to be lower than the polymer melting point. 3D printing materials such as ABS and PLA will already be deformed when the temperature is higher than 200°C. The ALD processes must therefore be compatible with rather low temperatures. Furthermore, as depicted by the review of Parsons and co-workers [64], the ALD precursors often infiltrate and react with polymeric substrates, which can alter the eventual 3D printed products. As shown by numerous studies, the risk of reaction between the polymer and the precursors increases with temperature and long exposures times. In addition, the presence of functional groups in the polymer chains also increases the potential infiltration of the ALD precursors [51, 52, 59, 64–68]. Thus, even if most of the ALD processes are compatible with the coating of 3D printed objects, these considerations must be taken into account and the processes have to be tuned accordingly to coat certain 3D printed materials. When the processes developed are compatible, the unique capability of ALD to coat complex objects with such control over the layer deposited, makes this route particularly relevant and attractive. This innovative combinatorial approach has been used for different and various applications, such as aerospace, photoelectrocatalysis, filtration, biomedicine, or solid-state batteries.

Kestila et al. combined polymeric additive manufacturing and an ALD-coating to produce satellite propulsion components with improved structural integrity and thermal resistance [69]. The components were made of two different polymers, namely acrylonitrile butadiene styrene (ABS) and polyamide, and were coated with alumina by ALD. The Al2O3 layer allowed to enhance the structural integrity for the polymeric restrictors and progressively smoothed out the PA surface improving the argon flow through the restrictor, which might be due to increased surface smoothness [69]. Heikinen et al. have recently shown that ALD of alumina on porous 3D printed ABS plastics permits to considerably lower their vacuum degassing. Nyman et al. have also confirmed the low outgassing of ABS, but also polyether ether ketone (PEEK), polycarbonate (PC), and nanodiamond-doped polylactide (ND-PLA) 3D printed materials with an Al2O3 ALD coating [70]. Thus, the combination of plastic 3D printing with ALD opens prospects for the fabrication of laboratory vacuum tools, and is also suited for spacecraft tools and in-space manufacturing applications [70, 71]. Moll et al. also coupled powder bed additive manufacturing with CVD and ALD of nitrides, to prepare 3D Ti-6Al-4 V structures highly resistant to hightemperature oxidizing environments. Coupling CVD and ALD on the 3D printed objects permitted to obtain thick coating and roughness reduction by CVD, and filling of narrow defects and reactivity mitigation by ALD [72].

Browne et al. employed additive manufacturing and ALD for photoelectrocatalysis, by depositing TiO2 onto 3D-printed electrodes. These electrodes were initially printed in inert stainless steel, and gained their catalytic functionality thanks to the ALD coating. The conformality allowed by ALD successfully permitted these 3D-printed electrodes to be used as photoanodes for water oxidation. The results presented have shown that the 3D-printed stainless steel electrode coated with ALD of TiO2 were considerably more active towards the water oxidation, and that the catalytic activity was enhanced by increasing the number of ALD cycles applied [73]. The team of Pumera et al. recently applied ALD to 3D-printed nanocarbon/polylactic acid electrodes to coat them with metal dichalcogenide MoS2 nanolayers [74]. The

MoS2 coated electrodes were then successfully applied for photoelectrocatalytic hydrogen evolution reaction (HER). Recently the group of M. Bechelany from the Institut Européen des Membranes (IEM, Montpellier, France) has developed in collaboration with the University of Zaragoza the functionalization of 3D printed ABS filters with MOF (Metal-Organic Framework) for toxic gas removal [75]. The fabrication approach at low temperature includes ALD of Zinc oxide on the ABS 3D printed filter followed by the hydrothermal conversion of ZnO to ZIF-8, Zeolitic Imidazolate Framework. The obtained filters show a good adsorption performance for dimethyl methylphosphonate, thus demonstrating their potential for toxic gas capture applications. Such types of 3D printed filters with an active MOF layer could have a wide range of applications in environmental fields such as adsorption systems for removing toxic gases or water pollutants.

In the biomedical field, the combinatorial approach has been applied to prepare silver-coated titanium orthopedic implants. [76] Using the selective laser melting (SLM) 3D-printing technique, titanium orthopedic implants have been fabricated with intricate geometries. The surface chemistry of the prepared implants has then been modified by coating them with a silver nanolayer by ALD. The inhibition of bacterial colonization obtained thanks to the silver coating resulted in the drastic reduction of the pathogenic biofilm. This result, combined with the increase of the vascularization and the osseointegration observed, opens a new path to this combinatorial approach for clinical orthopedic applications [76]. The "pure marriage" between 3D printing and ALD has also been exploited by Xue et al., who tailored the surface of 3D printed plastic earplugs using plasma-assisted ALD [75]. By combining 3D printing, plasma-assisted ALD and hydrothermal process, they loaded a layer of ZnO nanoarrays on the surface of the earplugs and thus improved the antibacterial properties of the earplugs, which enhanced the safety of the ear devices. In addition, they have shown that the sound insulation performances were higher than those of traditional earplugs. Finally, the field of solid-state batteries benefited as well from the combination of 3D printing and ALD. For example, thanks to an innovative 3D-printing ink formulation, a cell-based on a 3D-printed stacked array of LLZ (Li7La3Zr2O12, a solid lithium conductor) and lithium electrodes was fabricated, and ALD of alumina has been performed at the surface of the LLZ to allow the wetting of lithium [77, 78]. The ability to 3D-print solid electrolytes enables the manufacturing of unique ordered structures, and ALD permits their efficient functionalization, improving the overall efficiency of the battery device.

These few selected studies demonstrate the great potential of combining additive manufacturing and ALD. The combinatorial approach allows the fast prototyping of functional products with the additional precise control over their surface chemistry. As depicted in the presented examples, the benefits of combining 3D printing and ALD nanocoatings can be applied to many complex surfaces, and the lack of materials that can be 3D printed is at least partially solved by the use of ALD coatings. Thus, this novel approach allows synthesizing precisely integrated and customized architectures with tailored surface performance, and/or eventually the bulk properties of the materials thanks to ALI, paving the way towards innovative and functional products, and opening prospects for many potential applications.

## **5. Conclusion**

Although ALD was initially developed to exploit the possibility it offered to obtain continuous, pin-hole-free thin films even over large areas, in the last years there have been different approaches to perform ALD in a localized fashion, giving rise to the ASD field. As it has been discussed, these methods are based on different *Nanometric 3D Printing of Functional Materials by Atomic Layer Deposition DOI: http://dx.doi.org/10.5772/intechopen.101859*

approaches allowing either a high spatial resolution in XY (at the nanometer) or simpler more direct approaches that provide direct patterning at the millimeter and micrometer level in XY. In any case, and given these approaches are based on the ALD method, the control in Z is nanometric. The possibility to have spatial control over the ALD process can be exploited as a new gas-based technique for the 3D printing of functional materials at different scales, providing a unique approach to the fabrication of functional materials with complex shapes. Beyond using ALD as a 3D printing technique in itself, the possibility it offers to coat (even infiltrate) complex shapes in a highly controlled way and with a large amount of different materials is ideal to nanoengineer the properties of pieces obtained by standard 3D printing approaches, thus expanding the range of applications that can be achieved. ALD should thus experience an important penetration in the 3D printing field in the coming years.

## **Acknowledgements**

D.M.-R. acknowledges support from the European Union's Horizon 2020 FETOPEN-1-2016-2017 research and innovation program under Grant Agreement 801464, and through the Marie Curie Actions (FP7/2007-2013, Grant Agreement No. 63111). The Agence Nationale de la Recherche (ANR, France) is also acknowledged for funding via the programs ANR-16-CE05-0021 (DESPATCH) and ANR-20-CE09-0008 (ALD4MEM). The French National Research Agency (in the framework of the "Investissements d'avenir" program (No. ANR-15-IDEX-02) through the project Eco-SESA) is acknowledged for a PhD Grant.

## **Author details**

David Muñoz-Rojas1 \*, Matthieu Weber1 , Christophe Vallée2 , Chiara Crivello1 , Abderrahime Sekkat1 , Fidel Toldra-Reig1 and Mikhael Bechelany3

1 Univ. Grenoble Alpes, CNRS, Grenoble INP, LMGP, Grenoble, France

2 SUNY POLY, CNSE, Albany, New York, USA

3 Institut Européen des Membranes, IEM, UMR-5635, ENSCM, CNRS, Univ Montpellier, Montpellier Cedex 5, France

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

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## **Chapter 13**