In vitro research represents the starting point in biological and medical investigation, and it is conducted using components of an organism (tissues or cells) that have been isolated from their usual biological surroundings and used to emulate different aspects of human body functions; this permits a simpler, more rapid, detailed, and convenient analysis than what can be done with whole organisms (Johnson et al. 2016; Miller and Spence 2017). The myriad of in vitro experimental models offers a diverse variety of experimental approaches, which can provide empirical data potentially unobtainable from whole-animal studies, also reducing the probability of bias when animal usage tests or clinical trials are performed (Miller and Spence 2017).
In vitro studies were normally used to evaluate the effects and/or the mechanism of action of mechanical (such as hydrostatic pressure, mechanical compression, ultrasound, magnetic or electromagnetic fields) or chemical factors (cytokine, growth factors, adipokines, drugs, mineral elements, etc.) on cell or tissue morphology and metabolism (Cheleschi et al. 2015; Cheleschi et al. 2018; Cheleschi et al. 2019a; Cheleschi et al. 2019b; Collodel et al. 2013; Fioravanti et al. 2010; Kloesch et al. 2011; Marrazzo et al. 2019; Shams et al. 2018).
In vitro investigation plays a pivotal role to advance research into the physiopathology of a particular disease, and to help the development of potential therapeutic strategies (Johnson et al. 2016).
In recent years, lots of advancements have been made in the area of culture systems, which have enhanced functionality and stability of cells in vitro.
Tissue of human or animal origin, monodimensional or tridimensional cell cultures (for instance keratinocytes, chondrocytes, fibroblasts, polymorphonuclear cells), cell suspensions, or co-cultures can be employed. Studies on primary cells are the most used model for in vitro tests because they closely represent the tissue of origin, are not modified, and provide excellent culture systems for studying the physiological or pathological behavior of the native organ in vivo. Standardized and immortalized cell lines have been also developed to obtain indefinite subcultures with a high experimental repeatability and reproducibility, but without phenotypic characteristics of the in vivo tissue (Hanks et al. 1996; Miller and Spence 2017).
Each culture system presents advantages and disadvantages, and each is particularly suitable for exploring one specific aspect of cell metabolism, according to the aims of the study.
In the field of BT, tissue explants, primary cells, or immortalized lines can be used to investigate the potential biological effects of a single inorganic molecule (Carbajo and Maraver 2017; Viegas et al. 2019; Wallace and Wang 2015), or organic compounds (Gerencsér et al. 2019), or a mineral water as a whole (Fioravanti et al. 2011; Gálvez et al. 2018).
Among the inorganic molecules, which generally constitute the mineral waters, sulfur has currently been recognized as a crucial element with a wide range of functions, mainly when it was found in the form of hydrogen sulfide (H2S) (Carbajo and Maraver 2017; Viegas et al. 2019; Wallace and Wang 2015). H2S represents the main active molecule of sulfurous mineral-medicine waters; it is a small gaseous molecule traditionally considered as toxic gas, but, in the last years, scientific opinion has changed as more reports on its biological activity were published, and it is now considered a biologically relevant molecule (Carbajo and Maraver 2017; Wallace and Wang 2015).
H2S is an endogenous gasotransmitter, and, as such, it can be absorbed by numerous routes; it is able to penetrate the skin and mucosae and can therefore act at the cell level both in the skin and in internal organs (Burguera et al. 2017; Carbajo and Maraver 2017).
Its exogenous and endogenous donors at high micromolar concentrations were generally employed in in vitro research to mimic physiological functions of H2S at cellular level and to identify its potential mechanism of action (Carbajo and Maraver 2017; Wallace and Wang 2015).
Organic components of thermal waters were demonstrated to have biological effects contributing to the healing mechanisms, but their medical significance is not still fully understood (Varga 2012a). However, in a recent double-blind, randomized controlled trial on knee and hip OA patients, the organic fraction separated from the whole Szigetvár medicinal water and redissolved in tap water determined a major improvement of the studied clinical outcomes than what is observed in tap water alone (Hanzel et al. 2019). This finding explains the so-called Varga’s organic hypothesis, which supports that the biological effects of thermal waters are caused by bioactive organic molecules more likely than by the inorganic salt content (Varga 2010; Varga 2012b).
However, due to the raised number and variety of organics, the only method to analyze these organic mixtures, including possible interactions, is the determination of their biological activities in different tests. The most informative results were obtained from the Comet Assay (single-cell microgel electrophoresis for DNA damage) and the Salmonella Ames mutagenicity studies with the different chemical fractions of waters and peloids. Recently, (Varga et al. 2015)) presented a new application of the Salmonella TA strains originally engineered for the Ames mutagenicity test. After the organic extracts were isolated from five Hungarian thermal spa waters, the authors found that 4 of them showed a detectable UV-protective effect in Salmonella TA bacteria, demonstrating for the first time the UV-protective property of organic matter in natural thermal water samples.
However, it is plausible to think that the efficacy of a thermal mineral water is probably related to a complex relationship among a number of different chemical components (Fioravanti et al. 2011; Morer et al. 2017). This consideration leads us to identify the most suitable preclinical model to better investigate the mechanism of action of a mineral water as a whole.
In vitro studies on keratinocyte investigating the effects of BT on skin diseases
Gobbi et al. in 2009 and Mirandola et al. in 2011 (Gobbi et al. 2009; Mirandola et al. 2011) demonstrated the anti-inflammatory effect of exogenous source of H2S, natrium hydrogen sulfide, a fast-dissolving salt (NaHS), in normal skin–derived immortalized human keratinocyte cultures. After incubation of the cells with NaHS (400 mM, for 6, 12, 18, 24 h) in the presence of a specific mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) inhibitors, a reduced secretion of interleukin (IL)-8, IL-17, and IL-22, as well as a decreased cell proliferation and adhesion, through a downregulation of adhesion molecule expression, was observed, by reducing MAPK/ERK signaling phosphorylation (Table 1).
Furthermore, a group of investigators performed three different in vitro studies in order to evaluate the potential beneficial effects of Comano spa’s water (Trentino, Italy), a thermal hypotonic water containing various electrolytes as sodium, calcium, and bicarbonate, on the clinical manifestations of psoriasis (Chiarini et al. 2006a; Chiarini et al. 2006b; Dal Pra et al. 2007). Human psoriatic keratinocytes were incubated with different concentrations of Comano water (totally or partially dissolved in the culture medium) for an experimental period from 3 to 15 days and, then, the medium was collected and the cells processed for further analyses. The results demonstrated a reduced release and expression of vascular endothelial growth factor (VEGF) A, IL-8, IL-6, and cytokeratin (CK)-16 (a marker associated with keratinocyte psoriatic phenotype) in cells treated with all the studied concentrations of mineral water (Table 1).
Human keratinocyte cell lines, HaCaT, were used by Lee et al. (2012) to analyze the immunomodulatory or anti-inflammatory effects of a thermal spring water, from the Yong-gung oncheon (Ganghwa-gun, Korea), rich in sulfur, magnesium, calcium, and selenium, against the typical signs of inflammatory skin diseases. The Authors showed a suppressed expression of inflammatory cytokines (IL-6, IL-8) and an attenuated differentiation process of subsets of CD4+ T cells, into Th1, Th2, and Th17 cells, after 1, 4, 10, or 24 h of treatment with 50% concentration of spa spring water (Table 1).
HaCaT lines were also employed to an in vitro test of Bursa and Bolu waters, two traditional and historical thermal mineral waters of Turkey (Karagülle et al. 2018). Three days of incubation of the cells with 10% concentration of the two tested waters significantly reduced the gene expression of IL-1α, tumor necrosis factor (TNF)-α, and VEGF. The obtained results proved the anti-inflammatory and angiogenic properties of these spa waters in skin diseases such as rosacea and psoriasis (Table 1).
Recently, the effect of Hungarian Kakasszék spa (medicinal) water (KSZ) and Gyopáros spa water (GYP), containing a high variability of organic components, in preventing the adverse effects of solar or artificial UV radiation on the human skin was analyzed (Gerencsér et al. 2019). The organic fractions of each water were prepared using a procedure involving isolation of organics on Amberlite XAD macroreticular adsorbent resins. After 1 h incubation of HaCaT cells with organic-rich extract of KSZ or GYP with or without different timings of UV irradiation exposure, GYP isolate incubation resulted to be able to prevent DNA lesions of keratinocytes induced by UV exposure (Table 1).
In vitro studies on fibroblast-like synoviocytes, chondrocytes, and osteoblasts investigating the effects of BT on joint disorders
Fibroblast-like synoviocyte cultures, derived from RA and OA patients, and chondrocyte cell line C-28/I2 were employed by Kloesch et al. (2010; 2012a; b) to study the molecular mechanism of action of H2S, by using its exogenous source, NaHS. These cells constitutively expressed and secreted large quantities of IL-6 and IL-8. Furthermore, their treatment with different concentrations of NaHS (0.030–1 mM, for a maximum of 12 h) transiently reduced the constitutive expression of IL-6 and IL-8 and the activation of MAPK/ERK signaling, as well as those induced by IL-1β (5 ng/mL, 1 h) (Kloesch et al. 2010, 2012a, b). On the contrary, high concentration of NaHS (above 0.5 mM) demonstrated opposite effects both on the expression of cytokines and cyclooxygenase (COX)-2 and on the activation of MAPK/ERK proteins (Kloesch et al. 2012a, b). These results explained the beneficial effect of H2S on inflammatory processes involved in the pathophysiology of RA and OA, underling the importance of sulfur baths as possible therapeutic effect in such kind of diseases, taking into consideration H2S exposure in terms of timing and concentration (Table 2).
Growing evidence underlines the relevance of H2S and its exogenous sources as anti-inflammatory and anti-catabolic agents in human OA chondrocyte and synoviocyte cultures (Table 2).
In 2012, (Fox et al. 2012) studied the ability of human primary chondrocytes and mesenchymal progenitor cells to synthesize H2S in response to pro-inflammatory mediators stimulation (IL-1β, IL-6, TNF-α, and lipopolysaccharide (LPS)), and their response to the exogenous slow-releasing H2S source (GYY4137). Endogenous H2S produced by the cells and the treatment with different concentrations of GYY4137 (50–500 mol/L for 18 h) significantly reduced cell death and oxidant-induced mitochondrial dysfunction, caused by inflammatory cytokines, via protein kinase B (Akt)/phosphoinositide 3-kinase (PI3K)-dependent signaling.
Li et al. (2013) assessed the effect of GYY4137 (0.1–0.5 mM) on LPS (10 μg/mL)-caused release of inflammatory mediators from human arthritis synoviocytes and articular chondrocytes. After 18 h of treatment, GYY4137 demonstrated a prominent anti-inflammatory effect decreasing the production of nitrite (NO2−), prostaglandin E2 (PGE2), TNF-α, and IL-6 from both cell types, reducing the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and COX-2, and limiting nuclear factor (NF)-κB activation induced by LPS.
Equivalent results were obtained in a study on human OA chondrocytes stimulated with IL-1β, used as prototype pro-inflammatory cytokine to reproduce the “OA-like effect” (Burguera et al. 2014). The results of the research proved the ability of NaHS and GYY4137 (0.05–1 mM, for 24 or 48 h) to significantly limited nitric oxide (NO), PGE2, and IL-6 released by the cells and at protein level, as well as the gene expression of NOS2, COX-2, prostaglandin E synthase (PTGES), IL-6, and NF-κB nuclear translocation activated by IL-1β stimulus (5 ng/mL). Furthermore, these Authors firstly demonstrated the anti-catabolic activity of these compounds through the downregulation of metalloproteinase (MMP)-13 in the supernatant and at protein level.
These data were confirmed by Ha et al. (2015) in a study on human OA chondrocyte cultures treated with NaHS (0.06–1.5 mM) in the presence or not of IL-1β (10 ng/mL) stimulus for 24 h. The compound markedly reversed the effects of IL-1β on the gene expression of COX-2, MMP-13, and NOS and on their production in the supernatant. In addition, NaHS inhibited the activation of the ERK/IκBα/NF-κB pathway which was induced by IL-1β.
In the same year, Sieghart et al. (2015) investigated the effects NaHS (0.06–1 mmol/L), in OA fibroblast-like synoviocytes stimulated with IL-1β (10 ng/mL). The Authors observed that 1 h of NaHS treatment reduced spontaneous and IL-1β-induced secretion of IL-6, IL-8, and RANTES, the expression of MMP-2 and MMP-14, and the phosphorylation of several MAPK proteins. On the contrary, sulfide source increased the phosphorylation of pro-survival factor Akt1/2, suggesting the ability of H2S to partially antagonize IL-1β stimulation via selective manipulation of the MAPK and the PI3K/Akt pathways.
Later, a similar research was conducted on OA cartilage extracts co-cultured with IL-1β (5 ng/mL) and NaHS or GYY4137 (200 or 1000 μM) for 21 days (Vela-Anero et al. 2017). At the end of the treatment, the histological and immunohistochemical analyses of the samples demonstrated a reduction of catabolic processes and a stimulation of cell anabolism. Indeed, a decrease in glycosaminoglycan destruction and MMP-3 and MMP-13 production caused by IL-1β, in addition to an increased synthesis of collagen type II alpha 1 chain (Col2a1) and aggrecans, was observed in NaHS or GYY4137-treated cells.
All these findings provide new information about the anti-inflammatory, antioxidant, and anti-catabolic properties of H2S and of its exogenous sources in in vitro cultures. H2S seems to act as a chondroprotective agent by regulating relevant factors implicated in OA pathogenesis and progression, and counteracting IL-1β pro-inflammatory signals that lead to cartilage destruction.
Furthermore, in 2013, Fioravanti et al. (2013) studied the potential beneficial effect of Vetriolo thermal water (Trentino Alto Adige, Italy), a highly mineralized water, strongly acidic sulfate (SO4−), rich in calcium, magnesium, and iron, in human OA chondrocytes cultivated in the presence of IL-1β (5 ng/mL). To better appreciate the properties of mineral water, it has been tested at different concentrations (100%, 50%, 25%), directly dissolved in the culture medium. The Authors showed a significant survival recovery rate, a reduction in NO levels, and expression of iNOS, as well as an enhancement of morphological characteristics of the cells, altered by IL-1β, in chondrocytes treated with 50% and 25% Vetriolo thermal water; these data demonstrated the chondroprotective role of Vetriolo mineral water.
On bone-derived cells, only a limited number of in vitro studies were performed to investigate the properties of exogenous sources of H2S (Table 2).
Firstly, Xu et al. (2011) showed the proliferative, antioxidant, and anti-inflammatory effects of 4 h of treatment with H2S donor, NaHS (100 μM), in hydrogen peroxide (H2O2) (400 μM)-stimulated murine osteoblast-like cell line. The Authors observed an increased cell viability, cell proliferation (by enhancing alkaline phosphatase activity), and reduced apoptosis, caused by H2O2, after NaHS incubation. Furthermore, the H2S source increased superoxide dismutase activity, while it decreased reactive oxygen species (ROS) production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and NO and TNF-α release, probably via p38 and ERK1/2 MAPKs. These results were later confirmed by Lv et al. (2017), in an analogous experimental study, examining the effect of GYY4137 (100 μM) added at the culture medium for 4 h in the presence of H2O2 (400 μM).
These findings are in agreement with what is observed in a research performed on human differentiated osteoclasts (Gambari et al. 2014). After an incubation period ranging from 72 h to 6 days in presence of NaHS (50–300 μM), Gambari et al. (2014) found a significant dose-dependent decrease in osteoclast differentiation and intracellular ROS levels, and an upregulation of nuclear factor erythroid 2-related factor 2 (NRF2) activity, related to an increased transcription of the downstream antioxidant genes. Thus, the Authors suggested the key role of NRF2 as a possible mediator of inhibitory effects of NaHS.
In 2017, Liu et al. (2017) pretreated rat primary osteoblast cultures with 400 μmol/L NaHS for 30 min, followed by an incubation in DMEM, with high glucose concentration (HG), for 12 h, before the analysis of cell proliferation, apoptosis, and mineralization. NaHS significantly prevented osteoblast injury induced by HG, through decreasing the rate of cell proliferation, increasing the number of apoptotic cells, and blocking the HG-induced osteoblast mineralization inhibition, via activating ATP-sensitive potassium (KATP) channels.
In vitro studies on lymphocytes, neutrophils, and eosinophils investigating the effects of BT on immune response
The protective effects of H2S and of its exogenous sources on cellular immune response was firstly investigated by Rinaldi et al. (2006) in a study on purified human neutrophils, eosinophils, or lymphocytes which were treated with NaHS at concentrations ranging from 0.23 to 3.66 mM for 24 h. The Authors found an increased short-term survival of neutrophils delaying the onset of apoptosis, while no changes in lymphocytes or eosinophils were observed. The pro-survival effect of NaHS was due to its inhibitory activity on caspase-3 cleavage and p38/MAPK phosphorylation at the protein level (Table 3).
A similar experimental protocol was performed, 1 year later, by Mirandola et al. (2007) in human purified peripheral blood lymphocytes. The cells were incubated with different concentrations of NaHS (from 0.20 to 4.0 mM), for a time period of 24 h, to examine its role in regulating cell death and cytotoxicity, and its anti-inflammatory properties. At the end of the treatment, a dramatically decreased proliferation of surviving lymphocyte subsets, CD8+ T and NK cells, as well as a reduced IL-2 production, induced in response to mitogens, were observed (Table 3).
Also, Sulen et al. (2016) investigated the ability of H2S sources to regulated the activation of signaling transduction pathways implicated in immune response. Human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors were stimulated with NaHS at concentrations of 10, 100, or 1000 μM for 10 min, and the phosphorylation of p38/MAPK, NF-κB p65, AKT, and cAMP response element-binding protein (CREB) was analyzed with flow and mass cytometry. NaHS induced phosphorylation of p38, AKT, and CREB, but not NF-κB. These results provided a description of a NaHS-induced signal transduction pathway in human primary immune cells that may have relevance for the role of sulfides in inflammation (Table 3).
Furthermore, H2S donors were used to examine their role in mediating the immune response in inflammatory bowel diseases. At this regard, nanomolar levels of Na2S and GYY4137 (50–500 nM) were employed to treat primary mouse T lymphocytes (CD3+) and OT-II CD4+ T cells at time points of 4, 10, and 24 h to establish whether endogenous H2S production is required for T cell activation, in mediating inflammatory response in such kind of diseases (Miller et al. 2012). H2S donors enhanced T cell activation assessed by CD69 expression, IL-2 expression, and CD25 levels, with a maximum capacity at 300 nM. Besides, activation increased the capacity of T cells to synthesize endogenous amounts of H2S via increased expression of cystathionine γ-lyase and cystathionine β-synthase. These findings lead to define H2S as an endogenous and exogenous immunomodulatory molecule in T cells signal (Table 3).
The proliferative activity of H2S donors was also demonstrated in experiments carried out on peripheral blood lymphocytes isolated from patients with systemic lupus erythematosus (Han et al. 2013). Various concentrations of NaHS (0.25, 0.5, 1, 2, 4, and 8 mM) and GYY4137 (200, 400, 800, 1600 μM) were added to the culture medium of the cells for different time points in order to evaluate the cell viability, cell cycle distribution, and expression of proteins involved in pathological pathways regulating autoimmune response. H2S donors inhibited the abnormal activation and proliferation of lupus lymphocytes through the AKT/GSK3β pathway (Table 3).
Another in vitro study has been performed in activated human neutrophils isolated from blood of healthy donors, and treated with increasing amounts of the sulfurous thermal water of Acqui Terme, Piemonte, Italy, for 15 min (Braga et al. 2008). The cells were stimulated with N-formyl-methionyl-leucyl-phenylalanine and phorbol-12-myristate-13-acetate before and after incubation with sulfurous water, then the luminol-amplified chemiluminescence methodology was used to investigate ROS and reactive nitrogen species (RNS) release. The results showed that this mineral water significantly reduced the luminol-amplified chemiluminescence induced by the stimulus, on average from 0.94 to 15.5 μg/mL of HS (Table 3).
The same Authors performed a similar experiment treating neutrophils for 15 min with different concentrations of the above mentioned sulfurous water or NaHS (Braga et al. 2010); elastase release was evaluated by spectrofluorimetry, and elastolytic activity of the cells was determined by measuring the diameter of the area of elastinolysis on elastine-agarose gel plates. Sulfurous water, at concentrations ranging from 4.5 to 18 mg/mL, and NaHS, from 2.2 to 18 mg/mL, significantly inhibited elastase release but did not show any direct elastolytic activity. This finding revealed the possible contribution of sulfurous water in controlling the inflammatory processes of upper and lower airway diseases (Table 3).
In 2013, Prandelli et al. (2013) used human primary monocytes to test the beneficial effects of Sirmione thermal water (Lombardia, Italy), very rich in sodium chloride, bromide, and iodide, and of NaHS at concentration of 2.5 mM. Thermal water or NaHS was added to the culture medium of the cells for 24 h in the presence or not of 100 ng/mL of LPS, then the release of pro-inflammatory cytokines and the formation of ROS were evaluated. NaHS efficiently blocked the production of TNF-α, IL-1β, IL-6, IL-12, CXCL8, and CCL5 induced by LPS, and limited ROS formation and antioxidant enzymes activity; Sirmione water did not induced the same results, but only enhanced the release of IL-10, probably due to the low concentration of S-based compounds reached at its non-toxic dilution. The Authors attested the anti-inflammatory and antioxidant properties of S-based compounds against the main manifestation of chronic inflammatory and age-related illness (Table 3).