Macrophage Polarization Is Decisive for Chronic Bacterial Infection-Induced Carcinogenesis

*Mishi Wasson, Sonia Kapoor, Manoj Garg, Sandhya Singh and Hridayesh Prakash*

## **Abstract**

Macrophages are the special cells of the immune system and play both immunological and physiological role. One of the peculiar characteristics of macrophages is that they are double-edged and highly plastic component of immune system. Due to this characteristic, they are responsible for both progressions as well control of a variety of inflammatory, infectious and metabolic diseases and cancer. These are found in the body in three major phenotypes, which are known as M0 (also known as naïve); M1 (classically activated macrophages); and/or M2 (alternatively activated macrophages) at normal physiological conditions. We have been exploring macrophages in context of bacterial infection and previously demonstrated that M2 polarization of M1 effector alveolar macrophages during chronic/persistent *Chlamydia pneumonia*, *Mycobacterium tuberculosis* and *Helicobacter pylori* pathogens are decisive for the infection induced cancer development in host. Since chronic infection with these pathogens has been associated with adenocarcinoma, therefore, we feel that disruption of macrophage plasticity plays crucial role in the host for the development of cancer. On the basis of this, we propose that in such pathological conditions, management of M1/M2 imbalance is paramount for minimizing the risk of developing cancer by chronic and persistent infection.

**Keywords:** macrophages, immuno-epigenetics, metabolic programming, sterile inflammation, cancer

### **1. Introduction**

Recent studies have demonstrated that macrophages display high grade of phenotypic plasticity due to which they can both enhance and inhibit immune response. This phenotypical plasticity of macrophages enables them to contribute to pathogenesis of large variety of diseases as well as homeostasis mechanisms. Due to this characteristic, these cells are now known as double-edge component of immunity as well. Many studies have demonstrated that these cells can enhance the progression as well as control many infectious and tumor [1] diseases. Both peripheral and tissue macrophages together constitute the reticuloendothelium system which plays a major role in both sensing microbial antigens and their subsequent eradication [2]. Macrophages are recruited to the inflamed/infected

tissues, react to a variety of stimuli, and acquire either classical phenotype also known as M1 or alternative phenotype also known as (AAM, M2). Classically activated macrophages are immunostimulatory in nature and have Th1-orienting capacity while M2 are immunoregulatory in nature and have Th2 programming capacity [3]. The latter ones are anticipated to support the survival of various intracellular pathogens during persistency and believed to promote neoplastic transformation of infected tissue micromilieu (**Figure 1**). AAM accumulation in majority of adenocarcinoma (around 10% cases) confers poor prognosis during microbial persistency. Therefore in such abnormal pathological conditions, selective elimination of macrophages by ablating colony-stimulating factor 1(CSF-1) in LySMcre and op/op mouse model [4] or by the use of pharmacological drugs such as clodronate liposomes [5], which are among few possible modalities for mitigating macrophage-associated neoplasia. Within the frame of the above mentioned, this chapter will discuss various strategies to repolarize tumorassociated macrophages (TAM) during cancer development and uncover how selective activation of M1 macrophages could control infection-induced cancer but also existing anti-tumor immune therapies in both mouse and human model of tumors with special emphasis on gastric and lung tumors and inflammatory diseases like inflammatory bowel disease (IBD), which are responsible for global mortality. This may be achieved by targeting the major intracellular signaling component such as sphingolipids and Th2/Th17 responses, which promote M2 phenotype during persistent infection and potentially involve in the development of cancer.

#### **Figure 1.**

*Schematic representation of various approaches by which persistent infection with human pathogens disrupts functional plasticity of effector macrophages and promote cancer progression. The figure depicts how certain pathogens exploit various cellular and genetic mechanisms and promote M2 polarization of iNOS+ effector macrophages which are the special and double-edge component of the immune system. Phenotypic and functional polarization of effector macrophage is decisive event and anticipated to escort pathogens for neoplastic transformation of infected tissues during latent infections.*

*Macrophage Polarization Is Decisive for Chronic Bacterial Infection-Induced Carcinogenesis DOI: http://dx.doi.org/10.5772/intechopen.88171*

## **2. Pathogens disrupt macrophage plasticity and effector response during persistency**

Recent study has demonstrated that a bacterial product known as trabectedin is toxic to macrophages. This product inhibits NF-Y and KLF-2/4 which is important for the differentiation of macrophages in tumor micromilieu [6]. Similarly mitigating NF-κB, STAT3 and HIF-1 are involved in the activation of naïve macrophage to M1 effector phenotype and hold tremendous therapeutic option for modulating macrophages activation. Histopathological analysis of persistently infected lungs reveals the infiltration by specialized macrophage known as foamy macrophages. These are lipid-loaded macrophages and quite refractory in their nature. These macrophages behave more like AAM and are actively involved in the clearance of cellular debris and dead bacteria containing neutrophils and DC [7]. In some cases of coronary atheroma patients, these macrophages acquire phenotype similar to TAM (tumorassociated macrophages) and harbor dead bacteria in their endosome [8]. The presence of these macrophages thus promotes non-immunogenic inflammation which is similar to cancer-associated inflammation and supports opportunistic survival of deadly pathogens. Both phenotypic and functional polarizations of M1/M2 effector phenotype of macrophages are believed to be one of the prognostic factors contributing to the development of tumor during persistent/latent infections (**Figure 1**) in host. Once infiltrated in the infected lungs, these AAM/foamy macrophages potentially modify effector T cells and predispose them also as refractory which are otherwise proficient in the killing of infected cells. These macrophages secrete a plethora of cytokines/growth factors like VEGF-β, TGF-β, hypoxia-inducible factor, and sphingolipids which altogether contribute to neoplastic transformation of infected tissue. High gradient of VEGF and TGF-β promotes the differentiation of regulatory T cells [9] and inhibits the effector response of CD8+ T cells [10]. On the other hand, sphingolipids particularly S-1P/ceramide (either host or pathogen-derived) are known to promote mitophagy [11], M2 polarization of infiltrating M1, or naïve monocyte/macrophage populations [12]. In view of this, and to restore Th1 effector immune response during latent infection, reactivation of M1 effector phenotype of macrophage thus represents the most suitable therapeutic interventions. Apart from this, modulating the cytokine network also seems to be the most effective strategy for boosting immunity for the management of latent/persistent infections.

### **3.  Various pathogens adapt various strategies to this purpose which range from conferring resistance to the apoptosis [13], immune evasion [14], and metabolic programming of myeloid cells [15] as shown in **Figure 2**. Of these, conferring resistance and insensitivity for cell death in the infected cell seems to be one of the most fundamental processes. A range of bacterial pathogens like *Chlamydia trachomatis* (*C. tr*), *Chlamydia pneumonia* (*C. pn*), and *Helicobacter pylori* (*H. pylori*) which are associated with the pathogenesis of lung [16] and stomach cancer [17] respectively, exploit death and immune signaling for surviving in the hostile environment of antigen presenting (**Figure 2**) and effector cells. We [18, 19] and others [20] have demonstrated that *C. pn* and *C. tr* increase the stability of various endogenous regulators of apoptosis

**Figure 2.**

*Bacterial pathogens potentially exploit and interfere in various pathways in committed macrophage for subverting effector mechanisms during latency. Pathogenic bacteria interfere with various key signaling pathways which are important for the effector responses, e.g., recognition by receptors, uptake, and phagocytosis, lysosomal degradation, and alter signaling pathways and secretion of Th1 cytokines for establishing Th2 bias.*

proteins called IAP and inhibit the activity of both apoptotic and inflammatory Caspases 3, 8, and 11 during latent infection. Upregulation of CIAP2 and XIAP proteins [19] during acute and persistent/latent infection has shown the increase in noncanonical signaling of NF-κB which is a master transcription factor involved in both cell death inhibition and inflammatory programming and autophagy for Th1 effector response during infection. Our recent study has shown that *C. pneumonia* potentially interfere with M1 programming of infected macrophages [21] when stimulated with their cognate innate and inflammatory stimuli which is due to increased expression of HIF-1 and p38MAPK proteins [22] which are known to promote unfolded proteins response (UPR) in the infected macrophages [23] which in turn predispose macrophages refractory to immune stimulation. Within macrophages, mitochondria is potentially involved in the innate immune response of macrophages against a variety of successful intracellular pathogens mainly by flushing catatonic peptides like LL37, CAP 12, and CAP 18 to cytoplasmic compartment for efficient capture and killing of pathogen in mature phagolysosomes [24]. Recent studies have amply demonstrated that most of the opportunistic pathogens interfere in the mitochondrial physiology by promoting mitophagy which jeopardizes innate immune defense of macrophages against pathogens. In such cases, tweaking mitochondria by using Smac mimetic-based interventions holds promises in the management of persistent infection. Although we have recently demonstrated that Smac mimicry [21] is capable of mounting an efficient immune response against mild pathogens, it fails to do so against pathogenic microbes like *Leishmania donovani*. Since pathogenic microbes enhance the expression of p38MAPK/HIF-1 pathways [25] for sabotaging macrophages functions, therefore we feel that at the moment targeting p38MAPK/HIF-1 in conjunction to Smac mimetics would be paramount for controlling pathogenic microbes. This is a quite intriguing aspect of

*Macrophage Polarization Is Decisive for Chronic Bacterial Infection-Induced Carcinogenesis DOI: http://dx.doi.org/10.5772/intechopen.88171*

the field and needs further in-depth investigation. In the same line, many pathogens are known to interfere with autophagy process which is yet another potential mechanism which influences antigen presentation by APC to T cells for the clearance of pathogens. Many pathogens attack mTORc1 complex and disrupt the autophagosome apparatus [26, 27] and inhibit their presentation by APC to T cells for immune surveillance during latent phase. Therefore pharmacological tweaking of autophagy may offer one potential strategy for clearing dead pathogens effectively from the lung tissue. Irrespective of acute or persistent phase, all pathogens utilize host metabolism for their survival inside the host. During persistency, many pathogens consume carbohydrate and protein reservoir of infected macrophages and alter their metabolic rates. l-Tryptophan [28] and glucose are critical for macrophages, and their fluctuation largely dictates the effector response of infected macrophages as well as the fate of pathogen in APC/phagocytes. During persistent infection, *Chlamydia* sp. utilize cellular depot of l-tryptophan amino acid by activating IDO gene and metabolize it to l-kynurenine which impedes glycolysis [29] in the macrophages. M1 effector macrophages rely on cellular depot of glucose for both activation and differentiation into effector phenotype; therefore, during latent infection, pathogens disrupt in the glucose metabolism and promote hypoglycemia rendering them refractory for optimum defense against pathogens.

#### **4. Human pathogens promote epigenetic changes in macrophages during persistency**

At genome level, *C. tr* infection causes global hypoacetylation and hypermethylation of lysine residues on core histones which alter histone post-translational modifications which differ between acute and persistent infections. Upregulation of pH2AX (Ser139) and H3K9me3 which are hallmarks of DNA double-strand breaks (DSBs) and senescence-associated heterochromatin foci (SAHF), respectively, during *Chlamydophila trachomatis* [30] infection suggested teratogenic manifestation of *Chlamydia* persistency. This is largely due to increasing levels of reactive oxygen species (ROS) which is produced during latent infection. ROS contribute to DNA double-stranded breaks leading to persistent DNA damage, which in turn triggers SAHF formation in an ERK-dependent manner [30]. CPAF and CADD proteins from *Chlamydophila* pathogens are known to perturb host cell cycle machinery and inhibit recruitment of the DNA damage response proteins pATM and 53BP1 to damaged sites interfering with DNA repair mechanisms [30]. Despite impaired DNA repair, infected cells continue to proliferate which are in turn supported by enhanced oncogenic signals such as ERK, CyclinE, and SAHF. These changes altogether lead to the malignant transformation of infected tissue. Similarly, other pathogens like *Campylobacter rectus* [31], which is associated with oral cancer, downregulate Igf2 gene and enhance DNA methylation at its promoter which can be attributed to bacteria-mediated epigenetic modifications to the host genome. Other pathogens like *Salmonella enterica* serovar Typhi, which is one of the prognostic factors for the susceptibility for gallbladder carcinoma, exploit MAPK and AKT pathways [32] which initiate and sustain neoplastic transformation of infected host. Macrophages sense and trigger immune response against pathogens via TLR-linked signaling cascade. Under normal circumstance, almost all pathogens trigger TLR signaling pathways for activating macrophages; however, only few obligate intracellular pathogens, in hitherto, interfere with TLR signaling directly or indirectly and limit defense mechanisms [33] of effector macrophage like pattern of cytokines secretions, their uptake, and phagocytosis by macrophages. Although there are multiple ways how a pathogen can interfere with TLR signaling, so far TLR2/4 triggered

hypoxia and associated sterile inflammatory response, and/or TLR masking/ shedding mechanisms have been identified and proposed [34, 35]. *Yersinia enterocolitica* and *Candida albicans* are known to induce immunosuppression through TLR2-mediated IL-10 release and differentiation of T-helper cells to CD4+CD25+ regulatory T cells [36]. *Yersinia* species secrete a virulence (V) antigen, LcrV, which binds to CD14 and TLR2, trigger IL-10 secretion, and mediate immunosuppression. It has recently been shown that a particular residue in the N-terminal region of LcrV targets TLR2 and is required for altering IL-10 induction via TLR2 [37]. Likewise, *H. pylori* escapes from recognition by the TLRs due to the removal of phosphate groups from the 1′ to 4′ positions of lipid A in LPS, which confers low negative charge to this molecule and increases the chance of escaping TLR recognition. The recognition of non-LPS ligands by TLR2 leads to anti-inflammatory responses that are associated with IL-10 production [38]. Flagellin of *H. pylori* is one of the PAMP which potentially modifies the N-terminal recognition domain of TLR5 and helps in escaping the innate immune responses. Manipulation of amino acid 89–96 of the recognition domain of TLR5 results in low affinity to flagellin binding [39]. Under recurrent/latent infection state, TLR2-mediated signaling, hitherto, inhibits IFN-γ response and hijacks Th1 programming of macrophages. A pathogen like *M. avium* inhibits IFN-γ signaling in TLR2-dependent manner where it enhances the expression of dominant-negative STAT1b. Similarly, 19KD protein of *Mycobacterium tuberculosis* inhibits IFN-γ-induced expression of HLA-DR and FcγR1 expression on human macrophages [40]. In addition to the induction of anti-inflammatory signals by TLRs, certain pathogenic microbes have developed strategies to either block or avoid their recognition by TLRs and subsequent activation of the innate defense. According to one recent study, phospholipids and Ypk protein of *Treponema pallidum* interfere in TLRs (TLR3, TLR4, and TLR9) signaling [41] by blocking the function of LPS-binding protein and CD14. Several bacterial pathogens have altered specific PAMP structures to circumvent recognition by TLR4 or TLR5; pathogens, such as *Porphyromonas gingivalis* or *Leptospira*, which have specialized LPS structures that only interact with TLR2; likewise, in *Helicobacter pylori*, the flagellin [39, 42] is not appropriately recognized by TLR5, approving the survival of the bacteria without loss of virulence. Virulent strains of *Salmonella typhi* escape from their recognition by host PRR by various mechanisms, which predominately include modifying their lipid A by various mechanisms including deacylation, palmitoylation, and the addition of aminoarabinose [43]. Pathogens have evolved in several ways of avoiding NO-mediated killing that plays a central role in effector response in phagocytes. *Salmonella typhi* reside in a specialized membrane compartment called the *Salmonella*-containing vacuole (SCV), which is similar to inclusion in the case of *Chlamydia* sp. in macrophages, and use a T3SS called *Salmonella* pathogenicity island 2 (Spi2), which protects them from reactive nitrogen intermediates. Spi2-deficient strains of *S. typhi* get colonized in iNOS+ compartment efficiently [44] with the intracellular organisms in the SCV. Intracellular organisms have also developed mechanisms to detoxify NO-mediated effects. These include the ability to repair damage caused by reactive nitrogen intermediates and to detoxify these molecules. Pathogens have evolved the strategies of inhibiting iNOS activity which is the characteristic feature of M1 effector macrophages. Mucosal pathogen *Citrobacterro dentium* causes a marked reduction in the level of iNOS activity in macrophages [45]. There are many reported examples of bacterial pathogens altering inflammatory cytokines related to signaling. *Staphylococcus aureus* proteins A and M bind directly to the TNF-α receptor 1, on respiratory epithelium, which then potentiates a chemokine and cytokine cascade and subsequent disease [46]. Similarly, *Shigella flexneri*, through exploring type III effector, OspG, which is a protein kinase, activates ubiquitin-conjugated enzymes, thereby affecting

*Macrophage Polarization Is Decisive for Chronic Bacterial Infection-Induced Carcinogenesis DOI: http://dx.doi.org/10.5772/intechopen.88171*

phospho-ikBα degradation and subsequent NF-κB activation. Both *Chlamydia pneumonia* and *Chlamydia trachomatis* promote shedding of TNF receptor 1 by activating TACE activity and shunt TNF signaling though TNFR2 [47] and resist antibacterial and inflammatory action of TNF which is major component of effector macrophages.

### **5. Potential interventions for reactivating refractory macrophages for therapy outcome**

While the application of antibiotics is sufficient to control acute infection however during persistent infection, the outcome of treatment mostly remains refractory. This is due to increased density of refractory macrophages in various affected tissues which resists many therapies as seen in many similar diseases like cancer and metabolic disease which is mediated with tissue accumulation of type 2 or tumor-associated macrophages. It is now well accepted by medical community that increased densities of these macrophages are associated with poor prognosis in many infectious, tumor, and metabolic disease. In such conditions antibiotics and/or chemotherapy would require an additional regimen for effective treatment. During past decades, the growing evidence suggested that TAMs clearly play an important role in tumor progression, metastasis, and resistance to available chemotherapies by modulating the microenvironment inside the tumor mass as well as in the stoma. Therefore, it is important to reeducate or target the TAMs (M2-like) to antitumor M1-like macrophage phenotype for successful treatment of several human malignancies. In the remaining sections of this chapter, we have discussed various macrophage-specific and nonspecific interventions for reactivating refractory population of macrophages for improving existing therapies.

#### **5.1 Neoadjuvant for retuning refractory macrophages**

Many interventions have been made to reactivate or retune the TAM, but most of them could not influence the disease outcome profoundly. In this context our recent studies have shown neoadjuvant impact of low-dose radiation for retuning TAM, T cell-aided therapy [48], and subsequent normalization of vasculature in solid tumor-bearing animals. Since infection induced adenocarcinoma is manifested with high grade infiltration of foamy macrophages, which are like M2 TAM, therefore, on the basis of our tumor studies, we propose low dose gamma irradiation as one of the non-specific therapeutic interventions for the management of persistent infectioninduced tumor development.

#### **5.2 Nanomedicine as immune adjuvant for refractory macrophages**

Nanomedicine has emerged as one of the new modalities for reprogramming of both naïve as well as refractory macrophages toward their effector phenotype and thus represents one potential intervention for the management of latent infectious disease. We and others have recently demonstrated that due to their size and unspecific adjuvant properties, nanocarriers/nanocapsules can penetrate inflamed tissue microenvironment effectively and deliver drug in controlled and sustained rate for exerting adjuvant actions on macrophages in the inflamed and fibrotic lesions of infected tissue. Nanomedicine-based approaches may impact refractory macrophages at various levels, namely, (i) enhanced infiltration of fresh monocyte/macrophages, (ii) direct killing, and (iii) in situ polarization of AAM/foamy-like macrophages during chronic infection to assist clearing of infection. One of the interesting

mechanisms by which nanoparticle may improve the therapy outcome is to control the differentiation of naive monocytes toward iNOS+ M1 effector macrophages and replace CD11b+/iNOS-/Arginase-1+ AAM during chronic infections. In this context, our recent work has shown that a certain biodegradable amino acid-based pNAPA nanocapsule can potentially stimulate naïve macrophage to the M1 effector phenotype. On the basis of these merits, the nanocapsules may be used as adjuvant for activating innate immune system for the management of infectious diseases and cancer. Another potential application of nanoparticles is to deliver drugs or biopharmaceuticals for preventing differentiation of effector phenotype of macrophage to refectory. In this context one study has shown that delivery of CCR2 and CCR5 siRNA-loaded nanoparticles was able to reduce the recruitment of monocytes to inflamed tissue [49]. Nanocarrier-based approaches can be used for the direct killing of the refractory macrophages as well. For instance, liposomal formulations have been developed for the delivery of bisphosphonates such as zoledronates and clodronates. Subcutaneous/ orthotropic injections of these nanocarriers result in the depletion of AAM accompanied with impaired angiogenesis and reduction in metastasis. Nonspecific targeting is the major issue with nanocarriers which can be addressed by tagging these nanocapsules with specific ligands such as LyP1 and mannose receptors (e.g., CD206) which are highly expressed by TAM/AAM [50] for effective targeting of macrophages. PLA-PEG nanoparticles, cyclodextrin nanoparticles, and liposomal formulations have been developed for loading drug cargoes such as sunitinib, IL-12 plasmids, TGF-β inhibitors, and VEGF siRNA for reprogramming of refractory macrophages for skewing in situ Th1 effector immune response against latent infections [51–54].

#### **5.3 Immunotherapeutics are the next-generation treatment modalities**

One of the key characteristics of both AAM and TAM is to restrict Th1 immune response/T-cell programming by virtue of their releasing of Th2 cytokines and growth factors, which stimulate the neoplastic differentiation of inflamed fibroblast in tissue [55]. One of the major mechanisms by which these cells limit effector T-cell response is to engage programmed cell death ligands 1 and 2 (PD-L1, PD-L2) [56] which are expressed by the AAM/TAMs. Pulmonary infiltration of lipid rich foamy macrophages is a typical evidence of persistent infection-induced nonimmunogenic/sterile inflammatory immune response during persistent/latent *C. pn* and *M. tb* infections. Foamy macrophages are special kinds of AAMs which have poor antibacterial defense mechanisms and serve as carriers of many pathogens during dissemination. These macrophages inhibit Th1 programming of CD4/8+ T cells and promote Th2 bias by secreting IL-4 and IL-13 in infected tissue micromilieu and help these bacteria in immune escape. These macrophages are known to express PD-1L which after binding to PD-1 T cells drives anergy in T cells. Binding of PD-1L to PD-1 receptor triggers functional anergy in cytotoxic T lymphocytes (CTL) which are otherwise effective in eradicating persistently infected dead cells. Many pathogens exploit these pathways as a major immune evasion mechanism for securing their opportunistic survival. For example, *Chlamydia* sp., *H. pylori*, and *Leishmania donovani* pathogens are known to modulate the expression of PD-L1 on macrophages [57–59] for dumping adaptive immune responses. In such conditions, blocking PD-L1 pathway by monoclonal antibody against PD-1/PD-L1 has been found to be effective in restoring phagocytic potential of macrophages for dead cell clearance and subsequent control of tumor in mice models. For this study, it is anticipated that co-administration of antibody against PD-1/checkpoint inhibitors (CTLA4) along with antibiotics would be beneficial for the management of latent infectious diseases. In the same line, vascular endothelial growth factor (VEGF), transforming growth factor (TGF-β), fibroblast growth factor (FGF), and platelet-derived growth factor

*Macrophage Polarization Is Decisive for Chronic Bacterial Infection-Induced Carcinogenesis DOI: http://dx.doi.org/10.5772/intechopen.88171*

(PDGF), which are potentially secreted by AAM and promote sterile inflammation, also represent potential pharmacological targets for enhancing immunogenic death of cells during latent phase. Colony-stimulating factor 1 receptor (CSF1R) represents yet another promising target for therapeutic interventions because CSF1R signaling is crucial for differentiation, recruitment, and survival of TAMs [60].

#### **5.4 Antibody/small molecule inhibitor targeting polarization of refractory macrophages**

Intracellular pathogens, during both acute and latent infection, fiddle with various signaling pathways which range from receptor-associated cell death and innate immune signaling, antigen presentation, vesicular transport, and phagocytosis pathways. Although we have disused these in earlier section, here we will discuss the pharmacological and clinical significance of various approaches which may be decisive for mitigating cellular perturbations in the host for restoring immune defenses of macrophage during persistency. In this context, our recent study [21] has proposed that Smac mimetic (IAP-specific inhibitors)-based strategy has potential for enhancing immunogenic cell death of infected cells and reactivating refractory macrophages for improved clearance. Due to these virtues, several Smac mimetics have entered in the second-phase clinical trial against cancer, and we anticipate that the same is expected to help immune system for the management of persistent bacterial infection as well. Other than this, many pathogens exploit MAPK pathways [61] for their benefits and induce production of IL-10 cytokines in the macrophages which further inhibits T-cell programming mainly by promoting T-cell exhaustion [62]. Other than this, elevated levels of p38MAPK promote sterile/ anti-inflammatory response, which supports opportunistic survival of pathogen inside macrophages. Likewise, many pathogens exploit cAMP/PKA pathways and acquire Th2 bias during their persistency [63] for securing their survival; TNF-α is a major and key cytokine responsible for receptor-mediated killing of infected cells. We (unpublished data) and others have shown that many intracellular pathogens, during persistency, potentially target this cytokine and inactivate cell death pathways in TACE- or ADAM-dependent manner. Pathogens like *Chlamydia* sp. secrete CPAF, CADD, and hsp60 exerting TACE activity and cause shedding of TNFR1 [21], which actually induces cell death. Interestingly these proteins which are secreted by chlamydial pathogens require MAPK activation for efficient shedding of TNFR1 [64]. Therefore on the basis of the above observations, designing a suitable MAPK/phosphodiesterase 4 (PDE4) inhibitors [65] thus represents a compelling approach for controlling bacterial persistency and associated immune evade mechanism. Sphingolipids are yet another dual-specific cellular targets [66] of many pathogens for deviating Th1 effector immune response [67]. We have recently demonstrated that S1P/ceramide rheostat is an important parameter which can largely dictate whether pathogen would undergo persistency or not [66]. In this direction, we have recently demonstrated that the gain of S-1P during acute *M. tb* infection affords protective immunity in host for controlling pathogen burden; however, the same is anticipated to promote mycobacterial persistency and thus in such conditions, employment of sphingolipid-based inhibitors, in hitherto, would favor host for breaking persistency and induction of protective immune responses.

#### **5.5 Future perspectives: macrophage-based palliative strategies for tissue homeostasis post-antibiotic purging**

Successful therapy post-antibiotic treatment infection should normalize the tissue microenvironment and restore homeostasis. This could be achieved by chelating oxidative stress and remnants of inflammatory response for the replenishment of tissue mass, which normally gets lost during various therapeutic procedures. Management of M1/M2 imbalance is believed to be the key for minimizing the risk of having cancer by chronic and persistent infection with intracellular pathogens. In the clinics, this can be achieved by exchanging refractory populations of macrophage with effector ones which can control the sterile reactions and tumorigenesis. However, due to the pro-inflammatory nature of iNOS+ effector macrophages, this may elicit another sequence of destruction, which alone may not be beneficial. Therefore in such delicate conditions, co-administration of M1 macrophage with mesenchymal stem cell regenerative approach seems to be optimum for reconstituting the affected tissues and organs. The potential inclusion of macrophage-mesenchymal cell-based therapeutic intervention could be categorized under prospective palliative therapies for restoration of physiological function post-treatment.


*Article* **How Academics and the Public Experienced Immersive Virtual Reality for Geo-Education**

**Fabio L. Bonali 1,2,\*, Elena Russo 1, Fabio Vitello 3, Varvara Antoniou 4, Fabio Marchese 1,†, Luca Fallati 1, Valentina Bracchi 1, Noemi Corti 1, Alessandra Savini 1, Malcolm Whitworth 5, Kyriaki Drymoni 1, Federico Pasquaré Mariotto 6, Paraskevi Nomikou 4, Eva Sciacca 7, Sofia Bressan 1, Susanna Falsaperla 8, Danilo Reitano 8, Benjamin van Wyk de Vries 9, Mel Krokos 10, Giuliana Panieri 11, Mathew Alexander Stiller-Reeve 12,13, Giuseppe Vizzari 14, Ugo Becciani 7 and Alessandro Tibaldi 1,2**

**Abstract:** Immersive virtual reality can potentially open up interesting geological sites to students, academics and others who may not have had the opportunity to visit such sites previously. We study how users perceive the usefulness of an immersive virtual reality approach applied to Earth Sciences teaching and communication. During nine immersive virtual reality-based events held in 2018 and 2019 in various locations (Vienna in Austria, Milan and Catania in Italy, Santorini in Greece), a large number of visitors had the opportunity to navigate, in immersive mode, across geological landscapes reconstructed by cutting-edge, unmanned aerial system-based photogrammetry techniques. The reconstructed virtual geological environments are specifically chosen virtual geosites, from Santorini (Greece), the North Volcanic Zone (Iceland), and Mt. Etna (Italy). Following the user experiences, we collected 459 questionnaires, with a large spread in participant age and cultural background. We find that the majority of respondents would be willing to repeat the immersive virtual reality experience, and importantly, most of the students and Earth Science academics who took part in the navigation confirmed the usefulness of this approach for geo-education purposes.

**Citation:** Bonali, F.L.; Russo, E.; Vitello, F.; Antoniou, V.; Marchese, F.; Fallati, L.; Bracchi, V.; Corti, N.; Savini, A.; Whitworth, M.; et al. How Academics and the Public Experienced Immersive Virtual Reality for Geo-Education. *Geosciences* **2022**, *12*, 9. https:// doi.org/10.3390/geosciences 12010009

Academic Editors: Hara Drinia, Panagiotis Voudouris, Assimina Antonarakou and Jesus Martinez-Frias

Received: 26 November 2021 Accepted: 21 December 2021 Published: 24 December 2021

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

**Copyright:** © 2021 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/).

**Keywords:** immersive virtual reality; geology; photogrammetry; education; Iceland; Santorini; Etna

#### **1. Introduction**

Virtual reality (VR) is considered as a modern approach that provides 3D visualization in geological sciences, geoinformation for data collection and dissemination [1–3], as well as an immersive user experience [4,5]. Nowadays, VR landscapes can also rely on open or ad hoc-created geospatial datasets (e.g., [2]), including digital terrain and photogrammetryderived 3D models [6], as well as bathymetric data [4]. The latter are also provided in the form of virtual outcrops and virtual geosites, and are considered as a key strategy to overcome common difficulties among students, such as visualizing three-dimensional concepts on a two-dimensional medium (for instance, a book), or on an image-based virtual tour [7–9].

A first attempt to apply VR techniques to studying geomorphological processes was made by Hilde et al. [10] and Anderson [11], who applied 3D visualization techniques to picturing ocean bottom topography by reconstructing a 3D scenario and a 3D tour. Subsequently, Anderson [12] refined the first approximation approach of Hilde et al. [10] to produce three-dimensional models of Mars using Viking Orbiter images, to study an extended period of glaciation in the Elysium Region. Other VR approaches focused on organising the so-called virtual tours of key outcrops, geolocated in a GIS platform, navigated by students by visualizing and studying 2-D images [7–9] or 3D digital outcrop models [13]. This approach has been introduced to support teaching activity in Earth Sciences, to reduce teaching costs, and to improve student learning efficiency and increase learning interest [9].

In addition, virtual tours have been useful at different stages: as a digital review before field campaigns, as a tutorial review, and as a digital asset substitute for field site inspection [8]. A mixed approach proposed a number of 3D models to be explored similarly to a virtual tour, using a PC, a tablet or a Smartphone; for example, McCaffrey et al. [14] applied this approach to petroleum geoscience, whereas Pasquaré Mariotto et al. [3] combined the use of virtual geosites with a WebGIS Platform (https://arcg.is/1e4 erK0 (accessed on 26 November 2021)) to improve geoheritage communication.

VR applications have been increasingly used in recent years for geoscience education, scientific research, geoheritage communication and geotourism purposes; in fact, due to this approach, geological sites are made more accessible and available, including those that are dangerous or expensive to travel to, or have limited access. According to previous research efforts [3,15], this can be largely attributed to the possibility of making 3D models available as a free web resource, providing users with a number of virtual geological landscapes. These are the so-called virtual outcrops or virtual geosites, which are available to the public via web resources and represent, in 3D, geomorphological features and structures with photorealistic textures, based on photogrammetry techniques [3,6]. As such, virtual geosites can be an innovative tool for popularizing geoheritage in a broader audience by explaining ongoing, active geological and environmental processes [3,15,16]. Virtual geosites can also engage younger generations, who are particularly interested in highly interactive forms of communication and teaching [15]. An important advancement in the exploration of virtual outcrops and virtual geosites is the immersive virtual reality approach tailored by Tibaldi et al. [5], which has been developed in the framework of two research and innovation projects: namely the Italian Argo3D (https://argo3d.unimib.it/ (accessed on 26 November 2021)) and the EU Erasmus + 3DTeLC (http://3dtelc.lmv.uca.fr/ (accessed on 26 November 2021)) Projects.

In these projects, users can explore virtual geosites in the first person, thus experiencing the feeling of an immersive environment as if they were directly in the field. Furthermore, they can either walk or fly in photorealistic and photogrammetry-based landscapes (virtual scenarios) [6]. This possibility allows them to explore remote or inaccessible areas that

would not be accessible otherwise, e.g., when vertical cliffs are present or when exploring recent volcanically and tectonically active areas.

With the final aim of raising awareness about the above-cited projects, we organized nine educational events, with the total participation of 459 attendees, belonging to different age groups and cultural backgrounds, ranging from the lay public to highly-specialised scientific community members, and involving both students and researchers in Earth Sciences.

The participants firstly experienced immersive virtual reality and afterwards provided their feedback on the experience via anonymous questionnaires, the results of which are presented in this paper. Participants were categorised in the following five groups, four of which are subgroups:

For the dissemination events, we selected four Virtual Geosites which are considered as stunning volcano–tectonic environments due to their cultural, historical and scientific value. These are: the Metaxa Mine located in the Santorini volcanic complex in Greece [17]; a rift-transform triple-junction [18]; the 1984 Krafla eruption area [19], both located along the North Volcanic Zone (NVZ) of Iceland; and the Mt Pizzillo area, situated in the NE rift of Mt Etna, Italy [20].

In this paper, we explore the impact of immersive virtual reality as applied to geological exploration aimed at geo-education, geological exploration and, potentially, geotourism in volcanic areas.

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

#### *2.1. Immersive Virtual Reality Approach*

As detailed in Choi et al. [21], VR applications can be classified as non-immersive and, more importantly, fully immersive experiences. Non-immersive VR is typically referred to as 3D visualization and displays 3D models on a computer screen and/or mobile device without using any head-mounted displays. Fully immersive VR was applied during our dissemination activities and provided users with virtual ways to explore classical geological sites, being able to fly above virtual landscapes, using a suitable VR headset and thumbsticks. Users were provided with a holistic view of a virtual geosite, which allowed them to explore specific geomorphological and volcanotectonic features at a range of different aerial scales. This approach has been fully developed for Earth Sciences by Tibaldi et al. [5], with contributions from Gerloni et al. [22] and Krokos et al. [6]. The former has introduced a navigation mechanism dedicated to explore outcrops in a fully immersive way by replicating real-world field exploration. This approach is based on an offline-based visual discovery framework developed with the Unity game engine (https: //unity.com (accessed on 26 November 2021)). The application employs an Oculus Rift (https://www.oculus.com (accessed on 26 November 2021)) as a head-mounted VR device and allows earth scientists to navigate in their own immersive VR scenarios, based on photo-realistic photogrammetry outputs [6].

In our immersive virtual reality application, the landscape was built upon unmanned aerial system (UAS)-based photogrammetry techniques and is thus able to provide centimetric pixel size resolution for the resulting virtual landscape in the form of a 3D model. This has been successfully applied to models of different aerial extents and resolutions, ranging from about 50 to 1000 m (among the longest) and from 0.8 to 4 cm/pixel, respectively.

We used Agisoft Metashape (https://www.agisoft.com/ (accessed on 26 November 2021)) to process the 3D-model and generate a Wavefront OBJ Tiled model, which was designed and tested as the landscape input for our approach by Krokos et al. [6], Tibaldi et al. [6] and Antoniou et al. [17]. The use of 3D tiled models enabled us to obtain reliable representations of geometrical features especially when the objects are oblique to the studied outcrop.

When the user starts their virtual exploration, they walk around on a "solid ground surface" (Figure 1a–c), moving with the thumbsticks on the controllers. Our immersive virtual reality system allows them to choose between three different modes to navigate the scenery, named "walk mode", "drone mode" and "flight mode"(Figure 1b). In the default "walk mode", the user can navigate the virtual geosite slowly at a virtual height of around 2 m. (Figure 1c). In the "drone mode", the user can experience the site as a radio-controlled drone, flying above the virtual landscape. In the "flight mode" (Figure 1e), the user moves to a higher elevation and looks down over the terrain. This mode allows the user to move across the site at the fastest speed.

**Figure 1.** (**a**) User experiencing geological exploration. The VR headset and the input peripherals belong to the Oculus Rift. (**b**) The three exploration modes available in our application; (**c**) walk; (**d**) drone; and (**e**) Flight modes; road for scale.

#### *2.2. Dissemination Events*

The dissemination events described below were held between 2018 and 2019 in Italy, Greece, and Austria. Each participant was offered the possibility to explore two virtual geosites of their personal choice, each for five minutes, for a total of 10 min for each participant, during which they tried all three navigation modes.

During the virtual exploration, each user was supported by a trained staff member, who was knowledgeable about the virtual geosites and the use of the immersive tools. The aim was to offer a navigation tutorial to the user, so that they could quickly grasp the main geological and geomorphological features of the selected site. The dissemination events were as follows:

Communicating the science of geohazards in terrestrial and marine environments (http://3dtelc.lmv.uca.fr/). This event was held in Santorini (Thira), Greece (12–21 October 2018). On this occasion, we collected 14 feedback forms from participants aged 23–30.

#### *2.3. Questionnaires*

The aim of the questionnaires was to obtain feedback from people of different ages and backgrounds for the use of photorealistic immersive virtual reality in Earth Sciences, and to explore its applicability to teaching and studying as well as for communication purposes. The questions were categorised into three main groups: (i) general information and questions; (ii) general questions about the experience; (iii) specific questions about its use in Earth Sciences applications.

To facilitate the participants' replies, a specific procedure was designed. Firstly, the participants were asked to fill in an authorization form. For minors under the age of 18, parents were asked to fill in the authorization forms on their behalf. Once the required consent was given, the participants lined up to access the experience. Once their turn was announced, the participants were seated at the station equipped with a computer and the Oculus Rift (with headset and two controllers). In this phase, one of the staff members introduced the immersive virtual reality experience with a short tutorial to explain how

to explore the VR environment, as well as providing some basic geological notions about the environment in which they would soon be immersed. During each event, after the experience, the participants were invited to fill in a Google Form questionnaire that was the same for each dissemination event. The latter was divided into three macro categories:

As a final point regarding the questionnaires, it was decided to propose a limited number of questions to the participants, focusing on acquiring a large number of replies, following a trade-off approach. In fact, we planned to reach a reasonable balance between the possibility to acquire useful feedback about the use of immersive virtual reality for geo-education and the practical plausibility of administering the form within the events. We especially focused on getting feedback on the quality, usability, and usefulness of the developed system.

We did not carry out a specific analysis of perceived usability of the developed immersive VR environment and system, because it would have required the participants to fill in a very long and time-consuming questionnaire [23].

#### **3. The Virtual Geosites**

#### *3.1. The Metaxa Mine, Santorini (Greece)*

The Metaxa Mine is in the SE part of the Santorini volcanic complex, which is part of the active South Aegean Volcanic Arc. The volcanic centres extend from Sousaki and Methana (close to Central Greece) to the Nisyros-Kos islands (at the Eastern border with Turkey), with Santorini volcano being its southernmost expression. The latter is located on the Aegean microplate and is associated with recent volcano tectonic activity in the region [24]. The activity of the volcanic arc is related to the subduction of the African plate beneath the Eurasian plate [25], but locally around Santorini Island the stress field is extensional due to the slab rollback [26] and the associated regional tectonics.

The Metaxa Mine is an iconic geosite where visitors have a chance to view and explore the Late Bronze Age Minoan era deposits. Moreover, it is an industrial heritage site as the extraction of pumice during the previous century gave considerable income opportunities to the local population. Locally, the landscape presents an asymmetrical morphology [17], surrounded by almost vertical and overhanging cliffs and covered by debris flow deposits and remnants of anthropogenic activities. Its heterogeneous floor is scattered with small hills made of covered excavation materials. Along the vertical slopes of the mine, and especially at the entrance, an excellent outcrop showing Late Bronze Age deposits [5] can be observed. The virtual geosite for this site (Figure 2) has an extent of about 520 × 400 m and a texture resolution of 0.9 cm/pix. Further details are in Antoniou et al. [17] while

3D details with annotations can be found at https://geovires.unimib.it/ (accessed on 26 November 2021), within the geosites section, and at https://arcg.is/1e4erK0 (accessed on 26 November 2021).

**Figure 2.** 3D eastern view of the Virtual Geosite of the Metaxa Mine. See the white church on the right hand side for scale.

#### *3.2. 1984 Krafla Eruption Site, Northern Volcanic Zone (Iceland)*

The virtual geosite is located within the Krafla fissure swarm in the Northern Volcanic Zone of Iceland, along the emerging part of the Mid Atlantic ridge. This unique volcano tectonic terrain is composed of active rift zones, extension fractures, faults, eruptive fissures, and basaltic volcanoes. The area is occupied by a number of volcanic craters with a N-S alignment (Figure 3), lava flows, as well as a series of extension fractures and normal faults associated with the 1984 Krafla eruption [27]. The larger cone in the foreground (350 m × 150 m) was formed in 1984, at the end of the "Krafla Fires" eruptive cycle [28]. In its central part, one can observe the pyroclastic cone flank [19] as well as two normal faults which cross the post-Latest Glacial Maximum lava flows (older than 7 ka BP; 27). In the model, a cluster of recent monogenetic volcanoes (scoria cones) is also present, as well as an older scoria cone filled by the 1984 lava flow. The virtual geosite for this site (Figure 4) has an extent of about 1010 × 680 m and a texture resolution of 2.6 cm/pix. Additional details are made available in 3D with annotations at: https://geovires.unimib.it/ (accessed on 26 November 2021), within the volcano tectonics section.

**Figure 3.** 3D view of the virtual geosite of the Krafla eruption site; the scale is highlighted in the figure.

**Figure 4.** 3D view of the rift-transform triple junction virtual geosite; the scale is highlighted in the figure.

#### *3.3. The Rift-Transform Triple Junction, Northern Volcanic Zone (Iceland)*

To the east of Husavik, in Northern Iceland, a remarkable subaerial triple-junction intersection is preserved between the Husavik-Flatey Fault dextral transform fault and rifting in the Northern Volcanic Zone (Figure 4). The geomorphological and structural landforms are visible due to the presence of a sheet of pahoehoe lavas that record numerous structural features that are displayed in fantastic detail. These features include transform and extensional fault structures, riedel fault complexes, transpressional faulting and compressional strike-slip relay ramps, as well as second-order R, R' and P shears [18], providing a unique opportunity to investigate this dynamic structural environment using a virtual reality model created from drone imagery acquired over the triple junction region for the first time. Two interacting fault systems are clearly visible in the drone data and using the VR (the drone flight mode is particularly good for viewing these structures), comprising a

NW-SE transform-affinity faults of the Husavik-Flatey Fault and the roughly N-S normal faults of the Theistareykir rifting in the volcanic zone. The site is dominated by the vertical fault scarp face that extends north-south and the Husavik-Flatey Fault that extends away from this cliff face, as well as a series of small fault scarps with evidence of opening and right lateral movement.

#### *3.4. Mt Pizzillo Area, NE Rift of Mt. Etna (Italy)*

Mt. Etna is located along the eastern coast of Sicily, Italy, and since 2013 it has been inscribed on the UNESCO World Heritage list. Its long eruptive history, starting from 600,000 years ago [29] and its continuous gas and vapor emissions along with its sporadic Strombolian activity, has produced several spectacular lava fountains and lava flows which make this volcano an important reference for volcanological studies. Etna is, therefore, an ideal site to test immersive virtual reality and design a virtual survey of geological and structural features, such as its dense network of eruptive fissures forming the so-called NE, S and W Rifts. In particular, the area of investigation is located in the northern sector of the volcano: it belongs to the NE Rift, a group of eruptive fissures that mark a zone of frequent, shallow volcanic intrusions. The rough terrain is affected by active deformation (approximately 2 cm/yr [30]) and is covered by cinder cones and historical lava flows. The virtual geosite (Figure 5), which has an aerial extent of about 230 × 200 m and a texture resolution of 2 cm/pix, is an area located within Mt Etna's NE rift, on the northern flank of the volcano. Here, the users can observe a swarm of NE-SW extension fractures with centimetric dilation (up to about 70 cm) and several piercing points, which can be clearly observed along the fracture walls. The two widest main fractures gradually transit to normal faults, dipping towards each other. The faults can be clearly recognized on the flank of the pyroclastic cone of historical age [29]. The primary fracture located on the right is next to a short normal fault dipping to the left. By following the trace of the fault where it reaches the highest point on the flank, it is possible to see its partitioning into two faults that dip towards each other and form a graben structure. Further details are made available in 3D with annotations at: www.geovires.unimib.it, within the volcano tectonics section.

**Figure 5.** 3D view of the virtual geosite of Mt Pizzillo, along the NE Rift, Mt Etna.

#### **4. Feedback from the Users**

In this section we show the results acquired from 459 questionnaires from participants, aged 13–73. We focus both on the entire dataset of participants and on the following categories:

**Figure 6.** Graphs showing the results related to the General questions provided to the participants after their experience. To the left, relative frequency histogram displaying the age of the participants, grouped each 6 years, and, to the right, Pie chart showing the frequency of each category.

#### *4.1. General Questions*

The preliminary questions allowed us to identify the most frequent age groups that participated in the experience during our events. The histogram in Figure 6 shows that the users mainly belonged to two main groups: 13–31 years old, followed by the range between 31 and 49 years old, while older ages were less represented. For their backgrounds, the graph in Figure 6 shows that most of the users belonged to the lay public (38%), followed by academics (31%), school students (23%) and finally MSc class (age 21–30) (8%).

### 4.1.1.

As part of their feedback, all participants were asked about their familiarity with the VR equipment before the event. The results are displayed in Figure 7 and show that the majority of the users (54%) had not used similar equipment before. This observation, however, changes if we consider each category singularly. In particular, most students (52%) ranging from 13 to 20 years old (school students) had already used a VR headset before. On the other hand, lay public and academics show similar percentages: most (55% in the former case and 56% in the latter) had not used a VR headset before the dissemination event.

**Figure 7.** Bar charts showing the users' familiarity with VR equipment before the dissemination events. The results represent both the total number of participants and the background of each group, respectively.

#### 4.1.2.

Figure 8 shows the habits of the participants with respect to hiking/trekking: most users (72% of all participants) enjoyed the direct contact with nature and thus were used to going hiking or trekking. This percentage increased when considering both academics and MSc (84% in the first case and 83% in the latter). Finally, school students (59%) and lay public (68%) also reached quite high percentages. This was a fundamental question to compare replies from two different datasets of participants that were, or were not, addicted to field excursions, thus with different levels of expertise in landscape exploration.

**Figure 8.** Bar charts showing the habits of the participants with regard to hiking/trekking. The results represent both the total number of participants and the background of each group respectively.

#### *4.2. General Questions on Immersive Virtual Reality*

4.2.1. Which Is Your Favourite Navigation Mode?

For most participants, as displayed in Figure 9, the most popular navigation mode during immersive virtual reality exploration was the drone. This was clearly shown both by considering all participants (69%) and the single categories (for which the percentage ranged from 67% to 71%). Between the other two modes, the walk and flight ones, users mostly chose the walk mode (20–21%). A discrepancy, however, was found in the results provided by the MSc group, who showed a slight preference for the flight mode (17%) over the walk mode (14%).

**Figure 9.** Bar charts showing the preferred navigation mode. The results represent both the total number of participants and the background of each group respectively.

4.2.2. How Satisfied Are You with the Portrayal of the Virtual Landscape?

The users' satisfaction on the portrayal of the immersive VR environments showed that most declared themselves to be moderately (49%) to very satisfied (38%) (Figure 10), with the academics as the most satisfied (47%), followed by the lay public and MSc. The data for school students showed the lowest satisfaction rate for rank 5, even though the majority responded 4 and 5.

**Figure 10.** Bar charts showing the portrayal feedback of the Immersive Virtual Reality exploration. The results represent both the total number of participants and the background of each group respectively.

4.2.3. How Would You Rank Your Experience with Virtual Exploration?

More feedback was collected about the general satisfaction of the participants with the immersive virtual reality exploration. Most users were moderately to very satisfied with their experience (Figure 11). The highest percentages were shown by academics and the lay public. Based on our statistical analysis, school students were a little bit less satisfied when compared with the other categories. In fact, 18% declared themselves to be basically neutral.

**Figure 11.** Bar charts showing the experience of the participants during their immersive virtual reality exploration. The results represent both the total number of participants and the background of each group respectively.

#### 4.2.4. Would You like to Repeat the Experience?

Based on our results, a high percentage of the users wanted to repeat the experience (Figure 12). In fact, an average of 97% of all participants definitely wanted to repeat the experience; The school students category scored 100%.

**Figure 12.** Bar charts showing the participants' wish to repeat the immersive virtual reality exploration experience. The results represent both the total number of participants and the background of each group respectively.

4.2.5. Did You Experience Any Form of Physical Sickness during the Navigation? If so, Which One?

Short-term sickness was mentioned by 26% of the participants (Figure 13). This varied across the different groups, with academics being the most impacted, and MSc and school students being the least impacted. The overall 119 sickness effects reported consisted of: slight dizziness (65%), slight headache (22%), slight nausea (10%) and disorientation (3%). None resulted in an interruption of the experience, suggesting that these effects are minor in most cases. Regarding this point, we are aware that there are more thorough, systematic, and validated ways of evaluating the feeling of sickness due to the usage of VR technologies (in particular the Simulator Sickness Questionnaire—SSQ—introduced by Kennedy et al. [31]), but we only wanted to have an at-a-glance indication about the fact that some adverse effects were perceived by the users, and also to evaluate the real need of performing additional experiments on this point.

**Figure 13.** Bar charts showing possible side effects associated with the immersive virtual reality experience. The results represent both the total number of participants and the background of each group respectively.

#### *4.3. Teaching*

4.3.1. How Useful Do You Think Immersive Virtual Reality Is as a Studying/Learning Tool in Earth Sciences?

Figure 14 shows the effectiveness of immersive virtual reality as a learning tool. All participants believed that it could be very useful (59%) followed by moderately useful (30%). When considering each category, the perception of the users remained very similar. In particular, the lay public was found to be the most enthusiastic, as 67% found it 'very useful'. The other categories recognized the potential of this technology as a learning instrument. In particular, 56% of the school students and 57% of the MSc agreed with this scenario. Academics were less fond of it: 52% provided a very positive opinion, but 13% remained neutral.

**Figure 14.** Bar charts showing the possible efficiency of immersive virtual reality as a learning tool in Earth Sciences. The results represent both the total number of participants and the background of each group respectively.

4.3.2. How Useful Do You Think Immersive Virtual Reality Is as a Teaching Tool in Earth Sciences?

The great majority of the users (68%) declared that immersive virtual reality can be effectively used as a geo-education tool (Figure 15). In detail, academics and MSc showed similar results (70% and 69%, respectively) and found this possibility very promising. In contrast, school students only believed that it was 'very useful' at 52%, offering a general positive opinion when the 'moderately useful' replies, totalling 95%, were considered.

**Figure 15.** Chart bars showing the possible efficiency of immersive virtual reality as a geo-education tool. The results represent both the total number of participants and the background of each group respectively.

#### **5. Discussion**

Overall, the results of our initiative of using immersive virtual reality as a tool to carry out dissemination events on geo-education has been very positive. We held nine events, in three different countries, both in Italian and English languages, engaging a total of 459 international participants from various backgrounds. All dissemination events were carried out with the same format, virtual geosites and holistic view, aimed at understanding the impact of VR experiences on geosciences. Such events also involved people affected by temporal, or permanent, motor disabilities, who could explore "in person" inaccessible sites, proving how this technology could provide equality when dealing with geological field work and exploration. Thanks to the feedback given by all participants collected during these events, we provided a quantitative assessment of the immersive virtual reality experience, to evaluate its applicability as a tool for virtual geological exploration, with a particular focus on geo-education and communication. This represents an additional value, since previous attempts to evaluate VR techniques applied to Earth Sciences were mainly qualitative.

Generally speaking, there is a general agreement in the literature, mainly by practice and qualitative evaluations, that VR techniques are useful for teaching, as well as for learning in Earth Sciences (e.g., [7–9]). Over the last decade, several low-cost solutions have been made available to enable a quick and easy immersive virtual reality experience, both in terms of hardware and software solutions [32–34], providing teachers, students, and researchers with innovative tools to be applied in Earth Sciences. So far, in 2002, Kaiser et al. [32] applied immersive VR technology to improve mine planning, thanks to a collaboration among scientists with different areas of expertise. Recently, Kinsland and Borst [35] successfully used such technology to visualize geological data as 3D objects to be managed by scientists. As regards geo-education and research activity, McNamee

and Bogert [36] started using immersive virtual reality through a commercial application that allows users to fly and walk in a virtual scenario, in order to address two of the main problems in teaching geological processes in an introduction-level physical geology course: describing dimensions and scale. A slightly different approach has been used by Visneskie et al. [37], where the immersive experience was conducted with a set of 360-VR-Videos organized as a virtual tour to be used with mobile headsets. At a more advanced level, Tibaldi et al. [5], Antoniou et al. [17] and Caravaca et al. [38] designed a system where the user is capable of moving within a georeferenced and scaled 3D scene that can be explored simulating a real field trip.

Following this general view that VR tools are useful for geo-education and research activities in Earth Sciences, we aim to contribute to the discussion with a new relevant quantitative evaluation on the immersive virtual reality system developed by Tibaldi et al. [5] and many of the authors of the present paper, and successively applied for use in research activity by Antoniou et al. [17].

In evaluating the replies to the questionnaires, the first important output was that most participants would be glad to repeat the experience. This held for at least 97% of the respondents (all participants), with 100% positive replies provided by school students (Figure 12). More in detail, 96% of the participants, who had never used a VR headset before, would be willing to repeat the experience, whereas for those who had already tried a VR headset, the percentage rose to 98%.

For the exploration mode, most participants preferred to fly in "drone mode", around and above the main geological objects within the virtual geosites (Figure 9), by a percentage always greater than 67%, with the highest being reached by the lay public (71%), and a greater appreciation from participants that had already used VR (75%), when compared to those who had experienced it for the first time (64%). In addition, when considering if users were used to hiking or not, the drone mode reached a percentage of 68% and 71%, respectively, confirming that this was the best choice for navigation.

Another important aspect pertains to the type of virtual scenario we have proposed: again, in this case, the majority of participants ranked with high marks (4–5) the portrayal of the virtual landscape (87%), which is based on drone-captured pictures. This appreciation rate came especially from the lay public (86%), academics (89%), and school students (92%); on the other hand, the percentage of MSc was 71% (Figure 10).

The above results confirm the general appreciation for a photogrammetry-based virtual scenario. The results acquired from school students are the most interesting. Indeed, they show the lowest satisfaction for rank 5, even though the majority responded 4 and 5. We think that this could be due to the fact that school students are far more used to gaming and hyper real visualisations than other categories: as a consequence, their expectations might probably be higher. An improvement that can be made to the virtual landscape, for a better application to geo-education, is the possibility of adding everyday scaled objects to the scene, to help the user better perceive dimensions and distances. This approach could help overcome distance underestimation problems in VR compared to reality [39,40].

For the overall evaluation of the exploration of volcanic areas through the system, with the support of a geo-expert, the majority of the participants ranked with high value (4–5) the experience (87%), reaching a percentage of 93% among the lay public, 92% among academics, 88% among MSc, whereas the percentage plummeted to 72% for school students (Figure 11).

For the use of immersive virtual reality as a tool for studying and teaching Earth Sciences, it is worth noting that these questions have a different importance for academics than for both the student categories (MSc and school students). For the former group, both teaching and studying dimensions are relevant: in terms of studying activities, it is interpreted as an application to study landscapes, and thus as an application for research purposes as they are allowed to make direct observations. For MSc and school students, it is the studying/learning question that has a greater importance, since it would be applied as a tool for advanced learning activity. Regarding the results of these questions, as shown

in Figures 14 and 15, there is a general appreciation from all groups, with percentages in the range 85–96%: particularly, immersive virtual reality is considered as a great study instrument by 85–93% of the sample; the geo-education potential of it is highly regarded by 89–96% of the sample; in this latter case, the highest value (5) dominates the replies (Figure 15).

Some participants suffered from sickness effects, but none stopped the experience, and among those affected by sickness effects, only 8% of this subset would not like to repeat the experience, corresponding to about 2% of the total, suggesting that educational VR applications still need to be slightly improved. The latter point could be further explored through more detailed analyses on perceived adverse effects and system usability, following Vizzari [41]'s approach. It is noted that in order to achieve this, it is necessary to focus on a smaller number of participants, in ad hoc events lasting longer, with specific tasks being carried out, and that this is out of the scope of the present work. From a technical point of view, the use of VR headsets capable of working with a frame per second value higher than 90 can help reduce sickness effects [42]. In addition to that, it can be suggested that the user repeat the experience a few minutes after the first test. In fact, based on our personal experience, the more frequently immersive virtual reality systems are used, the more comfortable users become with the VR experience.

Overall, our results demonstrate and confirm the great potential of immersive virtual reality for geo-education and as a tool for geoscience communication to citizens, with possible applications for geotourism. Students, academics, and non-academics can explore virtual geosites in person, acquiring geological knowledge without temporal or spatial limitations, potentially reducing travel costs and carbon emissions [43], and decreasing unnecessary energy consumption.

The most important and innovative benefits we consider regarding the use of Immersive VR can be summed up as follows:

Furthermore, immersive virtual reality can be also used by students to examine the geology field route beforehand, so as to make the trip easier and more efficient. It allows users to carry out tasks that could be difficult in the real world due to constraints and restrictions, such as cost, scheduling, or location. Immersive virtual reality can be an alternative plan for field trip teaching in case the actual field trip cannot be conducted, or before scheduling a field trip to better plan the survey of the area.

We wish to highlight that both VR and immersive VR tools are especially crucial nowadays to overcome limitations due to travel restrictions and lockdown periods aimed at containing the COVID-19 pandemic, which have especially affected Earth Science teaching and research activities. In fact, teaching usually entails field activities with the involvement of large student groups and research activities often need field surveying in foreign countries. For both, the use of VR can also reduce the carbon imprint of education and research.

#### **6. Conclusions**

Our work has been focused on the quantitative evaluation of the participation of different types of people in immersive virtual reality experiences dedicated to Earth Sciences, especially volcanic areas. Our quantitative survey has involved a total of 459 questionnaires, filled in by participants at nine events between 2018 and 2019. The geological sites selected for the experience are located in Greece (Santorini), North Iceland and Italy (Mt. Etna).

The analysis of the questionnaires, based on nine questions, has enabled us to shed light on how useful this approach is for geo-education purposes. The majority of the respondents have shown a high degree of interest in the immersive experience. They particularly appreciated the opportunity to fly like a drone over the displayed geological objects. Another key outcome of the survey is the respondents' major satisfaction with the portrayal of the environment, made possible by cutting-edge, unmanned aerial system (UAS)-based photogrammetry techniques.

Importantly, 97% of the participants wished to repeat the experience.

With regard to the geo-education potential of this technique, most participants ranked with high value (4–5) the experience (94%), reaching a percentage of 96% among academics in Earth Sciences. Therefore, this study confirms that both students and academics see VR as a useful tool for geo-education.

Furthermore, our data shows the important role that immersive VR might have as a tool for:

Based on the above considerations, immersive VR can be also regarded as a groundbreaking tool to improve democratic ways to access information and experience, as well as to promote inclusivity and accessibility in geo-education while reducing travel, saving time, and carbon footprints.

**Author Contributions:** F.L.B. has tailored and managed all the events as well as organized and written the manuscript. E.R., F.V., V.A., F.M., L.F., V.B., N.C., A.S., M.W., K.D., F.P.M., P.N., E.S., S.B., S.F., D.R., B.v.W.d.V., M.K., G.P., M.A.S.-R., G.V., U.B. and A.T. have participated in the events and contributed to writing the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding devoted to this specific research.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The dataset with the replies from the participants is available online at https://doi.org/10.5281/zenodo.5792209 (accessed on 26 November 2021).

**Acknowledgments:** This research has been provided in the framework of the following projects: (i) the MIUR project ACPR15T4\_00098–Argo3D (http://argo3d.unimib.it/ (accessed on 26 November 2021)); (ii) 3DTeLC Erasmus + Project 2017-1-UK01-KA203-036719 (http://www.3dtelc.com (accessed on 26 November 2021)); (iii) EGU 2018 Public Engagement Grant (https://www.egu.eu/outreach/ peg/ (accessed on 26 November 2021)). Agisoft Metashape is acknowledged for photogrammetric data processing. This article is also an outcome of Project MIUR–Dipartimenti di Eccellenza 2018– 2022. Finally, this paper is an outcome of the Virtual Reality lab for Earth Sciences—GeoVires lab (https://geovires.unimib.it/ (accessed on 26 November 2021)). The work supports UNESCO IGCP 692 'Geoheritage for Resilience'.

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

#### **References**

### *Article*


*3.1. Characterization of Gum*

Table 1 showed physicochemical properties of CSG, FSG, and RSG. CSG consisted of 73.59% carbohydrate, 9.45% protein, 1.01% fat, 9.60% moisture, 6.26% ash (% *w*/*w*). FSG contained 78.56% carbohydrate, 11.38% protein, 2.16% fat, 9.08% moisture, 7.89% ash (% *w*/*w*) while RSG consist of 60.48% carbohydrate, 21.00% protein, 1.94% fat, 9.95% moisture, 6.63% ash (% *w*/*w*).

**Table 1.** Physicochemical properties of gum.

CSG: chia seed byproduct gum, FSG: flaxseed byproduct gum, RSG: rocket seed byproduct gum. The different lowercase letter in the same line indicates statistical significance (*p* < 0.05).

The protein content of gums is a very important parameter affecting the emulsification and foaming ability of gums. The protein content of the obtained gums was found to be higher than other natural and commercial gums [16,31]. RSG is rich in galactose and mannose, while FSG and CSG are rich in xylose and glucose. It can be said that RSG has a galactomannan structure as in many gums. The ratio of mannose to galactose was found to be 1.77 in RSG. The mannose to galactose ratio strongly affected the technological properties of gums such as their cold-water solubility, thickening, gelling, and crystalizing properties. The mannose galactose ratio was lower than guar gum (2:1) and LBG (4:1), and higher than fenugreek gum (1:1) [32,33]. In a similar study, xylose and glucose were reported to be higher than mannose and galactose from studies on the sugar profile of mucilage and gums obtained from flaxseed and chia seeds [34–37].

The FTIR spectrum of CSG, FSG, and RSG is illustrated in Figure 1. The results revealed that all gum samples showed a similar FTIR spectrum with some minor differences. This difference might be due to their protein content and different sugar and organic acid composition. Characteristic bands varying between 3500–3100 cm−1 are attributed to the hydroxyl (-OH) stretch that forms the gross structure of carbohydrates [38]. The bands between 3000–2800 cm−1 represent -C-H stretching of the aromatic rings and the methyl group (CH3) [38]. The bands at 1654 cm−1 and 1618 cm−1 for chia seed and its mucilage are assigned to a mannose ring [39].The bands at around 1597 cm−1 and 1422 cm−1 are due to the carboxyl groups of uronic acid residues in the gum polysaccharide or the presence of protein in the gum samples [40]. This result is compatible with the protein content of the gums. The wavenumber between 950 cm−1 and 1200 cm−1 is generally considered the fingerprint region of polysaccharides where the main chemical groups in polysaccharides are identified. The band at around 1030 cm−1 is assigned to C-O-C

stretching of 1→4 glycosidic bonds [41]. Also, the strong absorption at 1014 cm−1 shows the stretching vibration of the C–N [42]. The band at 864 cm−1 is assigned to the β-anomeric C-H deformation and glycosidic linkages of glucopyranose and xylopyranose units [38]. The FTIR spectrum obtained is very similar to many mucilage and gums studied in the literature. Hadad and Goli [43] observed the FTIR spectrum of flaxseed mucilage gave the absorption peaks at 3321, 2922, 1612, 1410, and 1050 cm−1. Darwish and El-Sohaimy [44] reported the absorption peaks of chia seed mucilage at 1739, 1539, 1444, 1419, 1157, 1058, and 618 cm−1.

**Figure 1.** FTIR spectra of the FSG (Flaxseed byproduct gum), CSG (Chia seed byproduct gum), and RSG (Rocket seed byproduct gum) in the spectral region between 400 and 4000 cm−1.

### *3.2. Flow Behavior Rheological Properties of the CSG, FSG, and RSG Solutions*

Figure 2 exhibits the flow properties of CSG (1.0, 1.5, and 2.0 %, *w*/*w*), FSG (1.0, 1.5, and 2.0 %, *w*/*w*), and RSG (1.0, 1.5, 2.0, 3.0, and 5.0 %, *w*/*w*) solutions with different concentrations over the range of shear rate from 0.01 to 100 s−1 at 25 ◦C. The decrease in viscosity by an increase in shear rate indicated a non-Newtonian shear-thinning (pseudoplastic) flow behavior for CSG, FSG, and RSG solutions at all concentrations. The decrease in the viscosity values of the samples due to the increasing shear rate can be explained by the breaking of the weak bonds between the molecules in the product as a result of the applied force and the weakening of the interaction between the components [45,46]. As predicted, the constant shear viscosity increased with the increase of gum concentrations. The most used hydrocolloids in the food industry, such as locust bean gum [47] and xanthan gum [48], showed shear-thinning rheological behavior. Similar shear-thinning flow behavior of gum dispersions was previously reported by Sanchez et al. [49], Marcotte et al. [50], Yamazaki et al. [51], Razavi et al. [52], and Chaharlang and Samavati [53]. CSG and FSG dispersions showed high viscosity and pronounced shear-thinning behavior at low concentrations (1–2%); however, more RSG exhibited weak pseudo-plasticity at lower concentrations. This might be explained by the varied origins of these gums. The gums with high molecular weight and strong intermolecular interactions have high viscosity solutions [54]. The orientation effect is another explanation for the shear thinning behavior. As the shear rate increases, the randomly distributed polymer molecules become more and more aligned in the flow direction, resulting in reduced contact between neighboring polymer chains [55].

**Figure 2.** Steady shear rheological properties of the FSG (flaxseed byproduct gum), CSG (chia seed byproduct gum), and RSG (rocket seed byproduct gum) solutions with different concentrations ((**a**) CSG (1–2%), (**b**) FSG (1–2%), (**c**) RSG (1–5%)).

Shear-thinning gums are commonly used to enhance the texture and rheological properties of food products. The high-shear processing operations, such as pumping and filling cause a reduction in the apparent viscosity of the solution. However, the high apparent viscosity produces a pleasant tongue feel during consumption [56].

The power-law model parameters (the consistency index (K) and the flow behavior index (n)) are shown in Table 2. The power-law model was successfully applied for the modeling of flow behavior properties of CSG, FSG, and RSG solutions (The coefficients of determination: R2 > 0.96). Many previous investigations have shown that the power-law model was the suitable model for describing the flow behavior of gum solutions [47,50,56,57].

**Table 2.** Steady shear power-law parameters of gum solutions.

CSG: chia seed byproduct gum, FSG: flaxseed byproduct gum, RSG: rocket seed byproduct gum. A different uppercase letter in the same column indicates statistical significance (*p* < 0.05).

The K values were found as 0.209–49.028 Pa·sn. The increase of gum concentrations increased K values for all types of gum solutions. At the same concentration, CSG had the highest K values, followed by FSG and RSG. The n values of CSG, FSG, and RSG solutions at all concentrations were less than 1, indicating that all gums showed a non-Newtonian shear-thinning behavior (Table 2). All the gum solutions had strong shear-thinning behavior with *n* values as low as 0.143–0.460, indicating a significant deviation from Newtonian behavior, and they, like many other shear-thinning hydrocolloids, have a high viscosity and pleasant mouthfeel at low shear rates. CSG, FSG, and RSG samples exhibited shearthinning behavior like locust bean gum [47], monoi gum [57], guar gum [58], and xanthan gum [48], which was explained to be due to weak bonds formed as a result of shearing [59]. When the n number is smaller than 0.6, it has been observed that non-Newtonian behavior becomes relevant [60]. The increase of gum concentrations caused decreasing n values. These results demonstrated that different concentrations of CSG, FSG, and RSG solutions affected the steady shear properties of CSG, FSG, and RSG solutions.

Figure 3 shows the viscosity of CSG, FSG, and RSG solutions with different concentrations over the range of shear rate from 0.01 to 100 s−1. As seen in Figure 2, the viscosity of the CSG, FSG, and RSG samples decreased with an increase in the shear rate at all concentrations due to the pseudoplastic behavior of gum solutions. The increase in shear rate led to a breakdown of molecular bonds, and therefore molecules became regular and internal friction decreased. As a result, the viscosity of CSG, FSG, and RSG solutions decreased. As the gum became entangled in the solution, the viscosity of the gum solutions increased with gum concentration, as can be seen in Figure 3. The higher viscosity value in CSG-2 (a solution containing 2% chia gum) compared to other gum solutions was associated with the strong interactions in hydrogen bonds [61].

**Figure 3.** The viscosity of CSG: chia seed byproduct gum, FSG: flaxseed byproduct gum, RSG: rocket seed byproduct gum solutions with different concentrations ((**a**) CSG (1–2%), (**b**) FSG (1–2%), (**c**) RSG (1–5%)).

CSG, FSG, and RSG solutions were sheared at a constant shear rate of 0.5 s−1 at 25 ◦C in the shear decay test. At 25 ◦C, Figure 4 depicts the change in shear stress of CSG, FSG, and RSG solutions as a function of time. Following that, the data were fitted to two different models: the Weltmann [62] model and the second-order structural model [63].

The computed parameters and their related determination coefficients are shown in Table 3. Both models could accurately reflect the time-dependency of shear stress values at all gum concentration levels with good R2 values.

**Figure 4.** Shear stress vs. time at a constant shear rate (0.5 s−1) for CSG: chia seed byproduct gum, FSG: flaxseed byproduct gum, RSG: rocket seed byproduct gum solutions with different concentrations ((**a**) CSG (1–2%), (**b**) FSG (1–2%), (**c**) RSG (1–5%)).

**Table 3.** Weltman and second-order structural kinetic model parameters defining the time-dependent flow behavior of gum solutions at 25 ◦C.

CSG: chia seed byproduct gum, FSG: flaxseed byproduct gum, RSG: rocket seed byproduct gum; constant shear rate (0.5 s−1). A different uppercase letter in the same column indicates statistical significance (*p* < 0.05).

The second-order structural model provides information on the change in timedependent flow characteristics caused by shearing, as well as the rate of breakdown, based on the sample's structured and nonstructured state. The rate constant (k) represents the rate of structural breakdown (thixotropy degree), whereas the initial-to-equilibrium viscosity ratio (*η*0/*ηe*) offers a relative assessment of structural breakdown quantity [26]. The parameters of the second-order structural model are shown in Table 3. Gum type also affected the magnitudes of k values and *η*0/*ηe* values. As can be expected, *η*0 and *ηe* values of gum solutions were increased with the increase of gum concentrations. Moreover, the k values and *η*0/*ηe* ratios of gum solutions were increased with the increase of gum concentrations. The highest k and *η*0/*ηe* values belong to CSG solutions, indicating that CSG solutions showed a faster rate of thixotropic breakdown and the extent of thixotropy.

The Weltman model predicted well the relationship between the shear stress and shearing time of gum solutions, and its parameters (A and B) were utilized to examine the effect of temperature on stress decay behavior (Table 3). Parameter A represents the shear stress threshold for structural breakdown, while the time coefficient-B represents the amount of structural breakdown caused by applied shear [64]. Negative B values indicate how soon the apparent viscosity achieves equilibrium [60]. Lower B values imply that the structure of a product is less affected by stirring. Therefore, RSG and FSG were affected less than CSG by stirring due to their lower B values. The Weltman model's coefficients were affected by the gum concentrations and applied constant shear rate [65].

### 3.3.2. Three Interval Thixotropic Time Test (3-ITT)

Figure 5 showed the structural recovery of CSG, FSG, and RSG solutions by 3-ITT, which simulates the sudden and nonlinear deformation of gum solutions. The structural recovery tendency of CSG, FSG, and RSG solutions increased with the increase of gum concentrations. The lowest concentrations of gum solutions caused the lowest structural recovery. These results showed that the structural breakdown observed in the second time interval due to high sudden shear force could be easily recovered as the gum concentration increased [66].

**Figure 5.** 3-ITT rheological properties of CSG: chia seed byproduct gum, FSG: flaxseed byproduct gum, RSG: rocket seed byproduct gum solutions with different concentrations ((**a**) CSG (1–2%), (**b**) FSG (1–2%), (**c**) RSG (1–5%)).

Table 4 indicates the structural deformation and recovery ratio determined by fitting 3-ITT rheological data using a second-order structural model. The thixotropic constant (k), initial storage modulus ( *G*0), equilibrium storage modulus ( *Ge*), and the ratio of *Ge* and *G*0 (*Ge*/*G*0) were calculated by the second-order structural kinetic model. The *Ge*/*G*0 value represents the recovery percentage; the larger it is numerically, the faster it can be evaluated to tend to recover. *Ge*/*G*0 values were between 1.124 and 1.750. CSG had the highest *Ge*, *G*0, and *Ge*/*G*0 values, indicating that CSG solutions were the higher recoverable character. As seen in Table 4, the k value indicates the thixotropic rate of samples, and a higher k indicates a higher recovery rate. For each type of gum, the higher gum content showed a higher k value. Samples containing the highest gum concentration showed the highest k and *Ge*/*G*0, indicating that the sample showed the highest thixotropic behavior and viscoelastic solid character. Also, FSG-2 had the highest k value.

**Table 4.** Second-order structural kinetic model parameters for 3-ITT.

CSG: chia seed byproduct gum, FSG: flaxseed byproduct gum, RSG: rocket seed byproduct gum. % D: % Deformation and % R: %Recovery. A different uppercase letter in the same column indicates statistical significance.

### *3.4. Viscoelastic Behavior of the CSG, FSG, and RSG Solutions*

A frequency sweep test was utilized for the determining viscoelastic behavior of gum solutions. Figure 6 indicated the dynamic viscoelastic characteristics of CSG, FSG, and RSG solutions with different concentrations. As seen, the changes in storage (G) and loss (G) values for gum solutions were demonstrated as a function of angular frequency (*ω*) at 25 ◦C. The structure of the gum solutions may be shown in dynamic mechanical spectra, which reveal the frequency dependence of storage modulus G and loss modulus G. There were no cross points between G and G values, revealing that G values were greater than G across the entire frequency range studied. As a result, gum solutions had typical gel-like behavior within the experimental frequency range at 25 ◦C. This viscoelastic behavior was in good agreement with that reported by Chaisawang and Suphantharika [67] and KUTLU et al. [68].

**Figure 6.** Viscoelastic behavior of the (**a**): CSG: chia seed byproduct gum (1–2%), (**b**): FSG: flaxseed byproduct gum (1–2%), (**c**): RSG: rocket seed byproduct gum (1–5%) solutions with different concentrations.

Non-linear regression was used to analyze experimental G and G values vs. *ω*, and the computed magnitudes of slopes (n and n), intercept (K and K), and coefficient of determination (R2) values are shown in Table 5. K values were determined to be higher than K values, indicating that gum solutions exhibited liquid-like characteristics. Similar results were reported in previous studies on different gum solutions [66–68] and different food products [69,70]. All of the solutions exhibited gel-like behavior due to the positive slopes (n and n > 0). The n value may be used to determine the strength and type of the gel; n = 0 indicates a covalent gel, while n > 0 indicates a physical gel. Low n values (around zero) indicate that G does not vary with frequency, but n values close to 1 indicate that the system acts like a viscous gel.

CSG: chia seed byproduct gum (1–2%), FSG: flaxseed byproduct gum (1–2%), RSG: rocket seed byproduct gum (1–5%); K and K: consistency coefficient (Pa·sn); n and n: flow behavior index values; R2: determination of coefficient. A different uppercase letter in the same column indicates statistical significance.

### *3.5. Rheological Properties, Zeta Potential, and Particle Size and Oxidative Stability of Low-Fat Vegan Mayonnaise Samples with Different Types of Gum*


**5. Conclusions**

**Author Contributions:** Data curation, Z.H.; Formal analysis, Z.H.; Funding acquisition, D.H.; Methodology, K.F.; Project administration, D.H.; Resources, W.S. Software, W.S. and Z.H.; Writing—original draft, Z.H.; Writing—review & editing, D.H. and K.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors are grateful for the financial support provided by the General Programme of the National Natural Science Foundation of China (Grant No. 51574032, 51674030).

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


3.2.2. Dry Test Tungsten Carbide on 100Cr6

The results of the tribological linear reciprocating ball-on-disc evaluation using a 100Cr6 disc and a tungsten carbide triboball for 50 mN and 200 mN load force are shown in Figure 5. The general comparison to the pairing 100Cr6/100Cr6 shows that the coefficient of friction is lower for all tested surfaces. On the polished reference surface, the coefficient of friction is 0.18 in the beginning and rises slightly up to 0.25 for a load force of 50 mN. With increasing load (Figure 5b), the COF increases to values of 0.55 at the end of the evaluation time of 500 s. The LSFL-structured surfaces lead to a COF increase over the entire evaluation time. For parallel and perpendicular movements, the COF starts at 0.45 and stays nearly constant for parallel sliding direction. The COF for the perpendicular movement on LSFL is higher (c.f. dry test 100Cr6/100Cr6) and rises to a steady state at 0.5. This behaviour also appears for the higher load force of 200 mN. The COF for LSFL-0 and LSFL-90 is initially 0.57 and increases to 0.6 for a parallel movement and to 0.68 for a perpendicular sliding direction. The LBIA surface introduce a small COF increase at the beginning of the test for both test loads. Using a perpendicular movement, the COF shows a time-based increase and runs from 0.24 to 0.3 for 50 mN and from 0.27 to 0.41 for 200 mN. Contrary to this behaviour, the parallel movement on the LBIA surface reveals a slight COF decrease with ongoing evaluation time. For 50 mN the COF decreases from 0.21 to 0.19. Using a load force of 200 mN, the COF reduces from 0.19 to 0.16.

**Figure 5.** Temporal evolvement of the coefficient of friction using tungsten carbide triboball without lubrication for a load force of 50 mN (**a**) and 200 mN (**b**).