**3. Results and Discussion**

## *3.1. Caracterization of Inorganic Ingredients*

#### 3.1.1. Particle Size

The granulometric distribution of PS9 and G30 is plotted in Figure 1. Both samples were unimodal and, therefore, homogeneous. PS9 showed to be finer than G30 (Table 4), the latter one slightly asymmetric. Calculated SPAN factors showed that the amplitude of particle size distribution for both samples had no significant difference (Table 4).


**Table 4.** Statistical particle diameters, SPAN factor (average ± s.d.; n = 3) and main modes of PS9 and G30 clay minerals.

In terms of hydrogels, the finer the particles of the solid phase, the higher the stability of the resultant semisolid system (no phase separation) and the better the textural properties (smoothness). Mineralogical composition of G30 (Table 1) included 26% *w*/*w* of quartz. The presence of this mineral could influence rheology and textural properties of hydrogels. Particularly, it infers an abrasive texture to the preparation if quartz particle sizes are big. Particle sizes higher than 150 μm could be abrasive, particularly when they have remarkable hardness, such in the case of quartz [66]. Despite the presence of 26% of quartz mineral in G30, its particles were smaller than 100 μm (Figure 1), which means that resultant hydrogels possessed smooth textures, which are crucial for the acceptance of the patients [67].

**Figure 1.** Particle size analysis of PS9 and G30. Differential analysis (up) and cumulative percentage of particles (down).

#### 3.1.2. Cation Exchange Capacity

The individual exchangeable elements and total CEC of PS9 and G30 are summarized in Table 5. Total CEC values were inside the expected CEC limits for sepiolite (9.18 mEq/100 g) and palygorskite (16.29 mEq/100 g) [68,69]. Sepiolite and palygorskite CEC are usually <25 mEq/100 g [70] with higher values usually related to impurities [71–81]. The higher CEC showed by G30 with respect to PS9 could be explained by the presence of 6% *w*/*w* of smectites and/or sepiolite in G30, as previously described.


**Table 5.** CEC results of PS9 and G30 (average mEq/100 g ± s.d.; n = 3).

The main exchangeable cations for both clays were Mg2<sup>+</sup> and Ca2<sup>+</sup>, which was in agreement with the chemical composition of PS9 and G30 showed by X-ray fluorescence analysis [63], though Na<sup>+</sup> and K<sup>+</sup> were also detected in small amounts. Na+, K+, Mg2<sup>+</sup> and Ca2<sup>+</sup> are essential elements since they are widely found inside and outside human cells [82–84]. The presence of suitable levels of ions such as calcium, magnesium, sodium and potassium in the wound bed are important to enhance the healing process. They allow the activity of the enzymes involved in the healing process, leading to the cascade of the repairing and regenerative processes. Specifically, calcium and magnesium levels should raise during the first 5 d of wound healing in order to promote granulation tissue formation and epidermal cell proliferation [85]. Moreover, during the restoration of the trans-epithelial potential of cells in the damaged tissue, Na+, K<sup>+</sup> and/or Ca2<sup>+</sup> play a crucial role [86]. The bioavailability of these cations in the wound site should promote the healing process and contribute to fasten the damaged area reparation.

Mg2<sup>+</sup> cations, abundant in both ALI and GR, have demonstrated remarkable properties for tissue regeneration and repair, particularly in the promotion of collagen formation and angiogenesis on skin wounds [56,87]. For this reason, it is conceivable that ALI and GR should perform wound healing effects, as previously remarked. The maximum exchangeable amount of Mg2<sup>+</sup> from PS9 and G30 corresponded to 17.67 and 37.14 mg/L, respectively (Table 5). Magnesium concentration due to the spring water was 5–12 mg/L, and this proved to be effective during wound healing by Sasaki et al. [56]. Consequently, both PS9 and G30 were considered as potentially effective minerals due to their exchangeable Mg2<sup>+</sup> content.

Normal homeostasis of mammalian skin is also maintained by elements such as calcium, modulating keratinocyte and fibroblast proliferation and differentiation [88]. Certain skin disorders, such as psoriasis, have been related to Ca2<sup>+</sup> disorders in keratinocytes [89,90]. Extracellular calcium is a determinant factor in the differentiation and maturation of fibroblasts, and its effectiveness is dose-dependent [89] Another recent study on wound healing demonstrated that calcium cations released from a calcium alginate wound dressing promoted endothelial cell growth and proliferation [91].

Zinc is also important during wound healing steps [85,88], though it was not detected as an exchangeable cation of PS9 nor G30 through the ICP-OES measurements performed. However, both ALI and GR waters contained Zn (Table 2).

Potassium has also demonstrated to favor wound healing (fibroblast differentiation, re-epithelialization, migration and proliferation of dermal cells), so its presence both in spring waters and hydrogels is considered as a positive feature [92,93].
