Fabrication and Evaluation of Electrospun Silk Fibroin/Halloysite Nanotube Biomaterials for Soft Tissue Regeneration.

The production of nanofibrous materials for soft tissue repair that resemble extracellular matrices (ECMs) is challenging. Electrospinning uniquely produces scaffolds resembling the ultrastructure of natural ECMs. Herein, electrospinning was used to fabricate Bombyx mori silk fibroin (SF) and SF/halloysite nanotube (HNT) composite scaffolds. Different HNT loadings were examined, but 1 wt% HNTs enhanced scaffold hydrophilicity and water uptake capacity without loss of mechanical strength. The inclusion of 1 wt% HNTs in SF scaffolds also increased the scaffold's thermal stability without altering the molecular structure of the SF, as revealed by thermogravimetric analyses and Fourier transform infrared spectroscopy (FTIR), respectively. SF/HNT 1 wt% composite scaffolds better supported the viability and spreading of 3T3 fibroblasts and the differentiation of C2C12 myoblasts into aligned myotubes. These scaffolds coated with decellularised ECM from 3T3 cells or primary human dermal fibroblasts (HDFs) supported the growth of primary human keratinocytes. However, SF/HNT 1 wt% composite scaffolds with HDF-derived ECM provided the best microenvironment, as on these, keratinocytes formed intact monolayers with an undifferentiated, basal cell phenotype. Our data indicate the merits of SF/HNT 1 wt% composite scaffolds for applications in soft tissue repair and the expansion of primary human keratinocytes for skin regeneration.

In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics.

The long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell's fate. We used ECM from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A2 decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that dermal fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epidermal sheets with a potential clinical use.