**1. Introduction**

Chronic wounds are a current health problem with devastating consequences for patients and contribute to major costs to healthcare systems and societies. This type of wound results from an impaired wound healing process and is usually characterized by prolonged or excessive inflammation, persistent infections and inability of the dermal and/or epidermal cells to respond to repair stimuli [1–3]. The USA total Medicare spending for all wound types has been estimated to range from \$28.1 to \$96.8 billion. Diabetic foot ulcers (one of the main chronic wounds) accounted for \$6.1 to \$18.7 billion [2], the main cost burden attributed to amputations [1]. The development and implementation of new wound healing management strategies and healthcare products is, therefore, imperative. In recent years, different technological strategies have been proposed, including clays, metals, polymers and lipid-based systems among others, as reviewed by Bernal-Chávez et al. and García-Villén et al. [4,5]. Particularly, clay-based dressings have been proven to be useful in wound healing [5–7]. Among the different clay-based formulations, those composed of a clay

suspended in mineral medicinal water, known as therapeutic muds, are widely used in clinical medical hydrology [8–10]. The solid phase of these systems is frequently incorporated into spring water to obtain a semisolid formulation known as "artificial thermal mud" [11–14]. Thermal muds have demonstrated their clinical effectiveness against dermatological affections such as psoriasis [15–18], atopic dermatitis, vitiligo [19,20], seborrheic dermatitis, fungal infections, eczema [18,21–23] and acne [24]. These clinical effects have been traditionally associated with the liquid phase. Avène and La Roche-Posay spring waters increase the fluidity of plasma membranes on cultured human skin fibroblasts [25–28] and have been useful in the management of chronic inflammatory skin diseases. La Roche-Posay spring water protected cultured human skin fibroblasts against lipid peroxidation induced by ultraviolet A and B radiation [29]. Boron and manganese-rich thermal waters are used for the treatment of ulcers and chronic wounds [30–34].

The influence of the thermal mud's solid phase in the resulting clinical efficacy has not been studied in depth. The inorganic solid phase of thermal muds is mainly composed of clay minerals [8,10,35–37]. The clay mineral presence in wound healing formulations is supported by their already demonstrated biocompatibility with different types of skin cells [38–40]. The combination of clay minerals with other ingredients, such as polymers, allows the formation of scaffolding materials. In these occasions, clay minerals not only improved the mechanical strength and functionality of the polymers, but they also acted as synergistic ingredients for wound healing [41–44]. Biocompatibility of clay minerals such as halloysite, montmorillonite, palygorskite, sepiolite and imogolite have been widely studied [45–55]. Sasaki et al. reported that Mg2<sup>+</sup> and Si4<sup>+</sup> ions released by a synthetic Mg-rich smectite clay mineral can promote collagen formation and angiogenesis on skin wounds [56].

Moreover, palygorskite ("attapulgite") has been used as scaffolding material when included in poly(lactic-co-glycolic acid) nanofibers, being crucial for mesenchymal cell adhesion and proliferation [57]. Sepiolite and palygorksite inhibit lipid peroxidation and possess anti-inflammatory properties by reducing neutrophil migration and edema [58,59]. Pharmaceutical grade palygorskite (Pharmasorb® colloidal) did not only demonstrate to be biocompatible but also to protect fibroblasts from carvacrol cytotoxicity [6].

As discussed, both spring waters and clay minerals have been separately studied as potential wound healing ingredients. Synergistic effects would be expectable when both ingredients are formulated as spring-water/clay hydrogels. The role of these systems in wound healing studies has been scarcely addressed. The existing studies include a clinical study of diabetic gangrenous wounds treated with volcanic deposits muds [60], black-mud Dead Sea effects in wounded mice [61] and wound healing activity of emulsions prepared with a Brazilian clay [62].

With these premises, spring water hydrogels have been recently formulated and characterized, including mineralogical and chemical composition as well as textural and thermal properties, as a first step in the design of pharmaceutical-grade systems [63]. The second step would involve the study and evaluation of their biocompatibility. In this regard, the simplest starting point would include in vitro biocompatibility studies over skin cells like fibroblasts and in vitro wound healing studies.

Particularly, the in vitro biocompatibility and cell gap motility (wound healing) properties of two selected Spanish medicinal waters (obtained from Graena and Alicún de las Torres thermal stations), two clay minerals (palygorskite and sepiolite) and their corresponding hydrogels were studied. Additionally, particle size distribution and zeta potential measures as well as cation exchange capacities of the solid phases were carried out. In vitro wound healing tests were also evaluated by analyzing fibroblast F-actin microfilament organization by means of phalloidin staining. To the best of the authors' knowledge, this is the first time that nanoclay/spring water mineral medicinal hydrogels have been evaluated in terms of in vitro cytotoxicity and wound healing.
