*4.3. Biocompatibility of ALIG30@20*

In vitro biocompatibility of clay minerals has been widely studied [15,96–99]. Some clay minerals have already been shown to have proliferating activity in cellular cultures, such as montmorillonite and halloysite [100,101]. Nonetheless, the induction of cellular proliferation by palygorskite clay mineral is a rare result [102]. This result leads us to hypothesize that if ALIG30@10 was biocompatible and able to induce fibroblast motility during in vitro wound healing [17], ALIG30@20, with proliferative activity, is also a promising formulation for wound healing treatments, especially during the proliferative stage. The different performance between these two hydrogels could be due to physicochemical differences of the systems. That is, different rheological behaviors as well as different chemical performances of both hydrogels could be the factors governing the biocompatibility results. Moreover, the present results could also be due to the combination of both physical and chemical performances of the formulations. Table A1 shows the theoretical amount of mobile element released in the fibroblast culture during MTT tests. These calculations have been made in order to correlate Franz cells results with those of MTT.

Mn has been reported as an active ingredient of spring waters with wound healing activity [29] This, together with the Mn released results in ALIG30@10 and ALIG30@20 (Table 2), leads us to propose manganese as one of the possible factors explaining the proliferative effect of ALIG30@20 versus ALIG30@10 (Figure 2).

Calcium and zinc have been demonstrated to actively participate in cellular growth, in particular the Zn:Ca ratio, which was demonstrated to increase Zn:Ca during cell proliferation and the decline Zn:Ca during the remodeling phase [20,21,103]. This is due to a redistribution of calcium within dermal cells during the wound healing cascade [104], which is dependent on certain trace elements such as zinc. In fact, extracellular calcium has been shown to stimulate DNA synthesis in cultured fibroblasts in the presence of Zn [105]. This has been mainly ascribed to the cofactor role of Zn in different enzymes involved in fibroblast growth. Moreover, Zn also plays an important role as a structural component of essential proteins. Some in vitro studies demonstrated that, even if proper growth factors and nutrients are present in the fibroblast culture medium, deficiencies of Zn translate to insufficient intracellular calcium and, ultimately, to impaired fibroblast proliferation [106,107]. From the release values of these elements, the Zn:Ca ratio of ALIG30@10 was 0.00465 and 0.01060 for ALIG30@20 (obtained from Table A1), which could be a significant factor inducing the proliferation of fibroblasts in ALIG30@20. It is also worth pointing out the fact that G30 showed a remarkable amount of Zn, thus being the ingredient providing this element. On the other hand, the major amount of Ca is provided by ALI. Any of the formulation ingredients on their own have been shown to induce cellular proliferation (see MTT results in García-Villén et al. [17] and Figure 2). This indicates that both ALI and G30, properly combined in a certain concentration, are necessary to induce fibroblast proliferation. Consequently, the proliferative effect is ascribed to the formulation itself, proving once again the major importance of formulative studies. By the same token, the Ca:Mg ratio also changes along the wound healing cascade. In fact, an increase in Mg levels is observed to favor cellular migration. Grzesiak and Pierschbacher stated that the Mg:Ca ratio was close to 1 during the migratory phase, and it reversed during the rest of the process [108]. ALIPS9 and ALIG30@10 hydrogels (aged for 1 month) showed Mg:Ca ratios (Table 2) closest to 1, which is in agreement with the induction of fibroblast migration already demonstrated for these formulations [17]. Nonetheless, the ALIG30@20 Mg:Ca ratio was significantly distant from this value, which happens during the proliferative phase.

The present results ultimately lead us to think that, apart from the amount of elements released from each hydrogel, their ratio and specific identity highly influence the final therapeutic performance of the formulation. Notwithstanding the fact that further studies are needed, it is noteworthy that the present formulations have the potential to be combined and administered at different times of the wound treatment by virtue of their chemical performance
