3.2.4. Cytocompatibility of Macrophages and Pro-Inflammatory Immune Response

Cytocompatibility was carried out by means of MTT assessment, recorded as intensity profile, analysed and presented on the macrophages in response to the scaffold components (a), the exposure and interaction with scaffolds after 24 and 72 h time points (b), as shown in Figure 11a,b. Moreover, TNFα concentrations (pg/mL), secreted by the macrophages after the contact with the components or the scaffolds are reported, in parallel to the MTT intensity readouts, in response to the components (Figure 11c); and the scaffolds (Figure 11d). From the collective results, it emerges that for all the cell substrates exposed to the components of the scaffold prepared in solution and for the LPS, at higher concentration (used as proinflammatory control) presented a full biocompatibility range when considering the two time points 24 or 72 h of contact with the macrophages (Figure 11a). Both HNT and MMT showed comparable biocompatibility although the macrophages' cytocompatibility decreased with MMT concentrations probably due to their different degradation profile up to 72 h.

**Figure 11.** Cytocompatibility of THP-1 macrophages of the scaffold components (**a**), and the scaffolds after 24 (plain color) and 72 h (oblique lines) of exposure and contact (**b**). TNFα cytokine expression and concentrations (pg/mL) for THP-1 cells exposed to components (**c**); and (**d**) scaffolds (mean values ± SD; n = 8) (GM: growth medium; LPS: lipopolysaccharide; P = pullulan; CS: chondroitin sulfate; CA: citric acid; CH: chitosan; HNT1: 1% w/w halloysite; HNT2: 2% w/w halloysite; HNT5: 5% w/w halloysite; MMT1: 1% w/w montmorillonite; MMT2: 2% w/w montmorillonite; MMT: 5% w/w montmorillonite; blank: unloaded scaffold; Val: valinomycin; Cys: cysplatinum) Statistics: \* = Mann–Whitney (Wilcoxon) W test *p* < 0.05.

The scaffold cytocompatibility towards fibroblasts and their proinflammatory response were evaluated only in the case of 2% clay mineral loading. In fact, the results obtained from mechanical properties and cell adhesion and proliferation capacity suggest that 2% clay mineral conferred to the scaffolds suitable stiffness/elasticity combined with the capability to support cell homing. After 24 and 72 h of growth, the scaffolds loaded with either HNT or MMT at 2% allowed macrophage viability similarly to that of their negative controls (GM, cell growth in standard conditions) (close to 75% of viability) (Figure 11b). However, the blank scaffold showed 50% cell viability with respect to the GM. The increase in the exposure time at 72 h also shows a decreased viability of the macrophages, when compared to their negative controls (GM).

Interestingly when looking at cytokine secretion, TNFα was secreted in significantly lower amounts from the cells in exposed and in contact with scaffold components for 24 h, while after 72 h of contact, the TNFα secretions were similar to the negative control, GM. The LPS positive controls provided the evidence that the cells were responsive to stimuli (proinflammatory agent) (Figure 11c). Considering 24 h exposure, the scaffolds caused a TNFα secretion similar to that of the GM. The scaffold loaded with HNT induced the TNFα secretion similar to those obtained when LPS was at a lower concentration but significantly lower than those observed for LPS at a higher concentration (Figure 11d). After 72 h of exposure time, all the scaffolds were assessed in their TNFα secretions that were not significantly different to the GM. This could be possibly also linked to the decrease in macrophage viability after 72 h in standard growth conditions. These results showed that HNT and MMT scaffolds did not show any significant proinflammatory activity compared to controls.
