Electrospun Silk Fibroin Scaffolds for Tissue Regeneration: Chemical, Structural, and Toxicological Implications of the Formic Acid-Silk Fibroin Interaction

The dissolution of Bombyx mori silk !broin (SF) !lms 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 nano!bers. 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 !mol/g. The greatest part was present as formyl ester, a small part (about 3%) as free FA. Approximately 17% of the 1,500 !mol/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 !bers 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 !broblasts did not show signi!cant 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

Goal 3 - Assicurare la salute e il benessere per tutti e per tutte le età

Goal dell’Agenda ONU 2030 costituiscono da tempo per PoliS-Lombardia il mainstreaming di tutte le attività: statistica, ricerca e formazione. Il Rapporto Lombardia – che legge lo stato del territorio regionale lombardo in ordine agli obiettivi e ai Target dell’Agenda ONU 2030 – è giunto alla sua quinta edizione, consolidando una linea di ricerca orientata alla sfida della sostenibilità, intesa in tutte le sue dimensioni (ambientale, sociale ed economica) e la sua complessità. Regione Lombardia ha fatto propria da tempo questa sfida, cogliendone appieno la portata; essa, infatti, non è stata lasciata a un singolo settore, ma è stata portata, giustamente, al centro della programmazione regionale

Learning curves.

<p>(A) Surgical preparation time (from heparin administration to flushing; R = 0.291, p = 0.397). (B) Procurement time (from flushing to harvest; R = 0.660, p = 0.0003); (C) Connection time (from harvest to lung connection to the circuit; R = 0.299, p = 0.370). Nonlinear regression analysis.</p

Ex Vivo Lung Perfusion in the Rat: Detailed Procedure and Videos

<div><p>Ex vivo lung perfusion (EVLP) is a promising procedure for evaluation, reconditioning, and treatment of marginal lungs before transplantation. Small animal models can contribute to improve clinical development of this technique and represent a substantial platform for bio-molecular investigations. However, to accomplish this purpose, EVLP models must sustain a prolonged reperfusion without pharmacological interventions. Currently available protocols only partly satisfy this need. The aim of the present research was accomplishment and optimization of a reproducible model for a protracted rat EVLP in the absence of anti-inflammatory treatment. A 180 min, uninjured and untreated perfusion was achieved through a stepwise implementation of the protocol. Flow rate, temperature, and tidal volume were gradually increased during the initial reperfusion phase to reduce hemodynamic and oxidative stress. Low flow rate combined with open atrium and protective ventilation strategy were applied to prevent lung damage. The videos enclosed show management of the most critical technical steps. The stability and reproducibility of the present procedure were confirmed by lung function evaluation and edema assessment. The meticulous description of the protocol provided in this paper can enable other laboratories to reproduce it effortlessly, supporting research in the EVLP field.</p></div

Instruments employed.

<p>Instruments employed.</p

Comparison of reconditioning phase settings in recently reported rat EVLP protocols.

<p>Comparison of reconditioning phase settings in recently reported rat EVLP protocols.</p

Changes over time in perfusion fluid parameters.

<p>Changes over time in perfusion fluid parameters.</p

Lung parameter changes during EVLP.

<p>(A) Pulmonary artery pressure (PAP). (B) Total pulmonary vascular resistance (TPVR). (C) Peak inspiratory pressure (Ppeak). (D) Dynamic compliance (Cdyn). Pressure recruitment maneuvers (RMs) were performed at 40 and 175 min. Results are expressed as the mean ± SEM (N = 10). One-way repeated measures ANOVA; p value was <0.001 for each parameter considered. Tukey’s multiple comparison test.</p

Protocol timeline.

<p>Schematic overview of EVLP protocol. Abbreviations: VT, tidal volume; RR, respiratory rate; PEEP, positive end expiratory pressure.</p