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Objectives: The optimal healing of skin wounds, deep burns, and chronic ulcers is an important clinical problem. Attempts to solve it have been driving the search for skin equivalents based on synthetic or natural polymers. Methods: Consistent with this endeavor, we used regenerated silk fibroin (SF) from Bombyx mori to produce a novel compound scaffold by welding a 3D carded/hydroentangled SF-microfiber-based nonwoven layer (C/H-3D-SFnw; to support dermis engineering) to an electrospun 2D SF nanofiber layer (ESFN; a basal lamina surrogate). Next, we assessed—via scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy, differential scanning calorimetry, mono- and co-cultures of HaCaT keratinocytes and adult human dermal fibroblasts (HDFs), dsDNA assays, exosome isolation, double-antibody arrays, and angiogenesis assays—whether the C/H-3D-SFnws/ESFNs would allow the reconstitution of a functional human skin analog in vitro. Results: Physical analyses proved that the C/H-3D-SFnws/ESFNs met the requirements for human soft-tissue-like implants. dsDNA assays revealed that co-cultures of HaCaTs (on the 2D ESFN surface) and HDFs (inside the 3D C/H-3D-SFnws) grew more intensely than did the respective monocultures. Double-antibody arrays showed that the CD9+/CD81+ exosomes isolated from the 14-day pooled growth media of HDF and/or HaCaT mono- or co-cultures conveyed 35 distinct angiogenic/growth factors (AGFs). However, versus monocultures’ exosomes, HaCaT/HDF co-cultures’ exosomes (i) transported larger amounts of 15 AGFs, i.e., PIGF, ANGPT-1, bFGF, Tie-2, Angiogenin, VEGF-A, VEGF-D, TIMP-1/-2, GRO-α/-β/-γ, IL-1β, IL-6, IL-8, MMP-9, and MCP-1, and (ii) significantly more strongly stimulated human dermal microvascular endothelial cells to migrate and assemble tubes/nodes in vitro. Conclusions: Our results showed that both cell–cell and cell–SF interactions boosted the exosomal release of AGFs from HaCaTs/HDFs co-cultured on C/H-3D-SFnws/ESFNs. Hence, such exosomes are an asset for prospective clinical applications as they advance cell growth and neoangiogenesis and consequently graft take and skin healing. Moreover, this new integument analog could be instrumental in preclinical and translational studies on human skin pathophysiology and regeneration.

The dissolution of Bombyx mori silk fibroin (SF) films in formic acid (FA) for the preparation of electrospinning dopes is widely exploited to produce electrospun SF scaffolds. The SILKBridge® nerve conduit is an example of medical device having in its wall structure an electrospun component produced from an FA spinning dope. Though highly volatile, residual FA remains trapped into the bulk of the SF nanofibers. The purpose of this work is to investigate the type and strength of the interaction between FA and SF in electrospun mats, to quantify its amount and to evaluate its possible toxicological impact on human health. The presence of residual FA in SF mats was detected by FTIR and Raman spectroscopy (new carbonyl peak at about 1,725 cm−1) and by solid state NMR, which revealed a new carbonyl signal at about 164.3 ppm, attributed to FA by isotopic 13C substitution. Changes occurred also in the spectral ranges of hydroxylated amino acids (Ser and Thr), demonstrating that FA interacted with SF by forming formyl esters. The total amount of FA was determined by HS-GC/MS analysis and accounted for 247 ± 20 μmol/g. The greatest part was present as formyl ester, a small part (about 3%) as free FA. Approximately 17% of the 1,500 μmol/g of hydroxy amino acids (Ser and Thr) theoretically available were involved in the formation of formyl esters. Treatment with alkali (Na2CO3) succeeded to remove the greatest part of FA, but not all. Alkali-treated electrospun SF mats underwent morphological, physical, and mechanical changes. The average diameter of the fibers increased from about 440 nm to about 480 nm, the mat shrunk, became stiffer (the modulus increased from about 5.5 MPa to about 7 MPa), and lost elasticity (the strain decreased from about 1 mm/mm to about 0.8 mm/mm). Biocompatibility studies with human adult dermal fibroblasts did not show significant difference in cell proliferation (313 ± 18 and 309 ± 23 cells/mm2 for untreated and alkali-treated SF mat, respectively) and metabolic activity. An in-depth evaluation of the possible toxicological impact of residual FA was made using the SILKBridge® nerve conduit as case study, following the provisions of the ISO 10993-1 standard. The Potential Patient Daily Intake, calculated from the total amount of FA determined by HS-GC/MS, was 2.4 mg/day and the Tolerable Exposure level was set to 35.4 mg/day. This allowed to obtain a value of the Margin of Safety of 15, indicating that the amount of FA left on SF mats after electrospinning does not raise concerns for human health.

Silk fibroin (SF) is an eligible biomaterial for the development of small caliber vascular grafts for substitution, repair, and regeneration of blood vessels. This study presents the properties of a newly designed multi-layered SF tubular scaffold for vascular grafting (SilkGraf). The wall architecture consists of two electrospun layers (inner and outer) and an intermediate textile layer. The latter was designed to confer high mechanical performance and resistance on the device, while electrospun layers allow enhancing its biomimicry properties and host's tissues integration. In vitro cell interaction studies performed with adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) demonstrated that the electrospun layers favor cell adhesion, survival, and growth. Once cultured in vitro on the SF scaffold the three cell types showed an active metabolism (consumption of glucose and glutamine, release of lactate), and proliferation for up to 20 days. HAAF cells grown on SF showed a significantly lower synthesis of type I procollagen than on polystyrene, meaning a lower fibrotic effect of the SF substrate. The cytokine and chemokine expression patterns were investigated to evaluate the cells' proliferative and pro-inflammatory attitude. Interestingly, no significant amounts of truly pro-inflammatory cytokines were secreted by any of the three cell types which exhibited a clearly proliferative profile. Good hemocompatibility was observed by complement activation, hemolysis, and hematology assays. Finally, the results of an in vivo preliminary pilot trial on minipig and sheep to assess the functional behavior of implanted SF-based vascular graft identified the sheep as the more apt animal model for next medium-to-long term preclinical trials.