*3.1. Polymeric Blend Characterization*

The characterization of the polymers in terms of conductivity (μS/cm), surface tension (N/m), and consistency (mN × mm) of all the polymeric blends is reported in Table 2.

The increase in clay mineral concentration caused an increase in conductivity, surface tension, and consistency. In particular, MMT at concentrations higher than 1% increased conductivity, surface tension, and consistency to values greater than those of the blank (blend without MMT). This behavior was less evident when HNT was blended with the polymeric mixture, probably due to the different particle sizes of the two clays. It is conceivable that the addition of HNT or MMT to the polymer blend caused a partial immobilization of the polymer chains due to charge–charge or hydrophobic interaction. In case of a lower amount of clay minerals added (1%), this determined a decrease in consistency and conductivity, while higher amounts (2% or 5%) had the opposite behavior probably due to an excess of charges from MMT or HNT not counterbalanced from the polymers in the solution [14].


**Table 2.** Conductivity, surface tension, and consistency of the polymer blends (blank) and polymer blends containing MMT or HNT at 1%, 2%, or 5% w/w (mean values ± SD; n = 3).

#### *3.2. Sca*ff*old Characterizations*

#### 3.2.1. Chemico-Physical Characterization

Advanced images such as SEM microphotographs of MMT- or HNT-based scaffolds and a blank scaffold in the dry or hydrated state are presented in Figure 2. The pore size evaluated for each scaffold is reported in an inset. Furthermore, in each image, the fiber diameter is reported.

In the dry state, the blank scaffold was characterized by fibers with a smooth surface, and fiber diameters with a coarse distribution around 1500 nm. HNT scaffolds were therefore characterized by the nanofibers' regular structure and smooth surface, where the addition of the clay minerals in the scaffolds provided a significant decrease in the fiber dimensions. The scaffolds containing 2% and 5% HNT had halved the diameter size compared to the blank scaffolds. Moreover, HNT determined a much more regular structure compared to the blank scaffolds. This was probably due to the unique structure of HNT, which are nanotubes with a high aspect ratio of 10 [9,20], thus capable of aligning along the fiber length and providing increasing surface tension. This allowed to obtain a more regular polymer solution jet during the electrospinning process. MMT scaffolds were characterized by nanofiber portions with a regular, smooth surface spaced out in a broaden interwoven, resembling knots, and with a wider structure organisation. These conceivably could be related to montmorillonite [9,21]. The increase of MMT concentration, especially in the 5% MMT scaffold, caused an increase in the surface roughness of the fibers, while the fiber diameters were significantly lower than those of the blank scaffold, although this was not influenced from the clay concentration. It is reported that clay minerals could act as a compatibilizer and this could positively affect the electrospinning of a polymer blend, containing positively and negatively charged polymers, as chitosan and chondroitin sulfate. Therefore, MMT or HNT could conceivably reduce the interfacial tension in the polymer blend, thus facilitating electrospinning, to obtain finer and more homogeneous nanofibers with respect to the blank [22]. Moreover, it is reported that the conductivity of the solution, influenced by the clay content, could increase the charge on the surface of the droplet to form a Taylor cone, and consequently could cause the decrease in the fiber diameter [23].

The presence of HNT or MMT in the scaffolds increased the systems porosity, and although there were not significant differences, the increase of clay mineral concentration increased the pore dimensions: this seems inversely related to the decrease of fiber dimensions. Porosity and fiber dimensions seem to have a crucial role for facilitating cell adhesion in the scaffold: The porosity could convert the scaffold from a surface to a fiber network, which could act as a sieve to the home cells.

The hydration significantly increased the fiber dimension, however no solubilization of the scaffold occurred thanks to the cross-linking by heating: The structural analysis (FTIR and SAXS) and the water holding capacity suggested that no new chemical bond was formed upon heating treatment while a polymer chain felting occurred when water was released due to thermal treatment, resulting in local physical multi-entanglement between the fibers, which could not be released by simple hydration. When HNT or MMT were at lower concentrations, up to 2%, the hydration did not alter the fibrous structure of the scaffolds, while when HNT or MMT were at a 5% concentration, the fibers were fused although the morphology was preserved. The higher content of hydrophilic clay minerals could weaken the overall scaffold structure, since the polymer chains in the matrix loosened their tightness causing a higher fiber swelling.

XRPD of all the scaffolds developed is presented as comparison to the pristine HNT and MMT, in Figure 3. A blank scaffold was characterized by an amorphous pattern and no crystalline or paracrystalline behavior could be detected. Pristine HNT was characterized by a peak at 12.25◦ 2θ corresponding to 7.24 Å, a typical height of the dehydrated HNT interlaminar spaces (Figure 3, peak labelled as #). The peaks at 20.14◦ 2θ and at 25.03◦ 2θ confirmed the HNT tubular structure and its phyllosilicate nature (Figure 3, peak labelled as ##) [24]. HNT-loaded scaffolds were characterized by patterns more similar to that of the blank scaffold rather than those of pristine HNT: In these patterns, only the peaks attributable to the phyllosilicate nature of HNT (peaks at 25.03◦ 2θ) were present and there was a signal increase directly related to HNT concentration. Since the diffraction angle remained constant in all the patterns and it was the same as in the pristine HNT, it could be argued that no enlargement of the interlaminar space of the rolled structure occurred.

**Figure 3.** Comparison of XRPD patterns of all the scaffolds developed against pristine HNT and MMT.

The pattern of pristine MMT was characterized by a peak at 7◦ 2θ due to the distance of the d001 basal reflection, corresponding to 12.2 Å, characteristic of predominantly Na+ smectites (Figure 3 peak labelled as +).

In the MMT scaffolds, the d001 basal reflection was shifted to approximately 6◦ 2θ. This corresponded to a distance of 14.0 Å, suggesting that there was an enlargement of the interlayer space (Figure 3, peak labelled as ++). This was associated with the intercalation of the biopolymer into MMT layers, probably as monolayer between the silicate layers [10,25].

FTIR spectra of all the scaffolds developed is presented as comparison to the pristine HNT and MMT in Figure 4. In the spectrum of the blank scaffold, the signals related to pullulan (P) and citric acid (CA) are marked. These characteristic signals were present also in all the scaffolds containing either MMT or HNT.

HNT spectrum was characterized by two signals at 3696 cm−<sup>1</sup> and 3622 cm<sup>−</sup>1, due to OH inner and outer stretching, respectively, while the MMT spectrum was characterized by a signal at 3624 cm−<sup>1</sup> caused by the Al–OH stretching. The characteristic peaks of both the clay minerals were hidden by a broad band due to a typical polysaccharide signal (hydrogen bonds of –OH and –NH2 groups). (pullulan: 3331 cm−<sup>1</sup> and chitosan: 3355 cm−1). Moreover, the vibrational band of NH3 <sup>+</sup> groups of chitosan could be identified at 1550 cm<sup>−</sup>1, as a shoulder [26,27].

**Figure 4.** Comparison of the FTIR spectra of all the scaffolds developed against to the pristine HNT and MMT. In the blank spectrum the signals related to P (pullulan) and CA (citric acid) are marked.

TGA (a,b) and DSC (c) profiles of all the scaffolds, compared to pristine HNT and MMT is reported in Figure 5. Thermal analysis was performed to characterize the role of the clay minerals in the scaffold structure. TGA and DCS profiles suggested than both HNT and MMT had high thermal stability.

TGA analysis suggested that all the scaffolds, independently of the clay mineral loaded and its concentration, were subjected to a slight weight loss corresponding to the evaporation of hydration water. This accounted for about 7% of the scaffolds weight (30–101 ◦C) (Figure 5a). DSC analysis (Figure 5b,c) showed a slight endothermic event between 30 and 110 ◦C, confirming the TGA results.

Additionally, characterization showed that all the scaffolds reported a more prominent weight loss (onset: about 230 ◦C; offset: about 400 ◦C) with greater mass loss to reach 26%, 12%, and 7% of residual weight for the MMT 5, MMT2, and MMT1, respectively. These coincided with two endothermic events in the DSC thermograms and these could be conceivably caused by the decomposition [28,29].

The clay minerals loaded into the scaffolds, independently from the types and concentrations used, maintained their thermal stability and were able to slightly stabilize the scaffolds towards thermal degradation, increasing the onset temperatures of each thermal event; this is particularly evident in the TGA profiles (Figure 5a,b).

The residual mass was related to the clay mineral concentration in each scaffold: HNT: 5.56% for 1% loading; 11.40% for 2%, and 21.02% for 5%; MMT: 5.46% for 1% loading; 11.02% for 2%, and 22.36% for 5%.

HRTEM microphotographs of the broadened parts of the fibers is presented in Figure 6, and Figure 7 reports their EDX spectra.

The HRTEM and EDX analysis evidenced that the broadened parts were based on clay mineral particles: The tubular structure of HNT and laminar one of MMT could be identified. Moreover, the elemental analysis showed the presence of Al and Si typical in the case of HNT and the presence of Al, Si, and Mg in the case of MMT, but also of S and C, to indicate that the inorganic material was embedded into the organic component.

**Figure 5.** Comparison of TGA (**a**,**b**) and DSC (**c**) profiles of all the scaffolds against pristine HNT and MMT.

**Figure 6.** HRTEM microphotographs of the broadened parts of the fibers.

**Figure 7.** EDX spectra of the broadened parts of the fibers.
