**1. Introduction**

The skin is the largest organ of the body and plays a pivotal role in maintaining physiological homeostasis against fluid imbalance, thermal dysregulation, and infections. It is formed by the epidermis, consisting of keratinocytes, and by the dermis, mainly based on the extracellular matrix (ECM) (collagen, elastin, glycosaminoglycans) and sparse fibroblasts. Damage or loss of integrity of the skin caused by a wound, may impair skin functions, exposing the body to potentially challenging situations. Wound healing is a complex event based on overlapping but well-orchestrated cellular and molecular processes to repair damaged tissue and restore skin function. Healing is divided in different phases (hemostasis, inflammatory, proliferative, and remodeling) and is accomplished by ECM molecules, soluble mediators, as cytokines and growth factors, various resident cells, and infiltrating leucocytes [1].

Acute wounds are mainly traumatic or surgical and generally heal within few weeks without any significant interventions, whereas in chronic wounds, the healing process remains frozen in the inflammatory state. These, commonly defined as wounds that fail to proceed through an orderly and timely process to restore skin anatomical and functional integrity. These include venous leg ulcers, arterial ulcers, diabetic ulcers, and pressure ulcers, such as bed sores. The increase in life

expectancy and the increasing prevalence of diabetic disease and venous insufficiency lead to an increase in chronic wounds. Although assessing the prevalence of chronic wounds is problematic because of the disparities in study design and their evaluation, they have become a major challenge to healthcare systems worldwide. It is estimated that the prevalence of ulcers ranges from 1% in the adult population to 3–5% in the over 65 years population; whereas globally it accounts for the 3–5.5% of the total healthcare expenditure as recently estimated [2,3].

Recently, clay minerals have been proposed in the biomedical field in tissue engineering as enhancers of cell attachment, proliferation and differentiation [4,5], and also as antimicrobials [6]. In particular, montmorillonite (MMT, Mx (Al2-yMgy) Si4O10 (OH)2 nH2O) and halloysite (HNT, Al2O3 2SiO2 2H2O) have been recently described as biocompatible and as proliferation enhancers [7]. Both MMT and HNT are phyllosilicates, having a planar and rolled structure, respectively. Nanocomposites, based on these biomaterials, have been developed to tune cell adhesion and their biocompatibility [8–11]. The cell interaction with clays remain unclear and not fully understood, and only a few recent studies have attempted to shed light on this interaction [7,12].

However, there are many evidences in literature related to the combination of nanostructured materials with nanoscale fabrication processes, to achieve high levels of morphological control, surface and mechanical properties [13–15]. In light of these, layered silicates are characterized by a high aspect ratio with the ability to confer high strength to 3D structures. Moreover, unlike most inorganic fillers, layered silicates are hydrophilic, and capable to interact with the polymer matrix, by changing surface tension, conductivity, and shear viscosity. Thus, the combination of clay minerals and nanofibrous scaffolds should lead to 3D architectures which facilitate cell homing, but also enhance the cell attachment and proliferation thanks to the enhancing properties provided by biopolymers. In particular, chitosan and chondroitin sulfate are polysaccharides capable to aid cell proliferation, and moreover, the latter is also providing protection against growth factor degradation by electrostatic interaction [16]. Having previously assessed the scaffold composition in the basic biopolymers [16]; these made of pullulan, chitosan, and chondroitin sulfate; the manufacturing processing was then addressed by means of electrospinning in a one-pot process to obtain a nanofibrous scaffold which proved to enhance the mechanical properties of the scaffold.

Given the advances achieved on these materials and their manufacturing processes, the aim of this work was the design, development and characterization of electrospun 3D scaffolds, entirely based on biopolymers, loaded with MMT or HNT, as a dermal substitute for skin reparation and regeneration tested in a preclinical model, leading to tissue reparation towards a complete skin restore.
