NDP-MSH treatment recovers marginal lungs during ex vivo lung perfusion (EVLP).

The increasing use of marginal lungs for transplantation encourages novel approaches to improve graft quality. Melanocortins and their receptors (MCRs) exert multiple beneficial effects in pulmonary inflammation. We tested the idea that treatment with the synthetic α-melanocyte-stimulating hormone analogue [Nle4,D-Phe7]-α-MSH (NDP-MSH) during ex vivo lung perfusion (EVLP) could exert positive influences in lungs exposed to different injuries. Rats were assigned to one of the following protocols (N = 10 each): 1) ischemia/reperfusion (IR) or 2) cardiac death (CD) followed by ex vivo perfusion. NDP-MSH treatment was performed in five rats of each protocol before lung procurement and during EVLP. Pulmonary function and perfusate concentration of gases, electrolytes, metabolites, nitric-oxide, mediators, and cells were assessed throughout EVLP. ATP content and specific MCR expression were investigated in perfused lungs and in biopsies collected from rats in resting conditions (Native, N = 5). NDP-MSH reduced the release of inflammatory mediators in perfusates of both the IR and the CD groups. Treatment was likewise associated with a lesser amount of leukocytes (IR: p = 0.034; CD: p = 0.002) and reduced lactate production (IR: p = 0.010; CD: p = 0.008). In lungs exposed to IR injury, the NDP-MSH group showed increased ATP content (p = 0.040) compared to controls. In CD lungs, a significant improvement of vascular (p = 0.002) and airway (Ppeak: p < 0.001, compliance: p < 0.050, pO2: p < 0.001) parameters was observed. Finally, the expression of MC1R and MC5R was detected in both native and ex vivo-perfused lungs. The results indicate that NDP-MSH administration preserves lung function through broad positive effects on multiple pathways and suggest that exploitation of the melanocortin system during EVLP could improve reconditioning of marginal lungs before transplantation.

Preclinical Validation of SilkBridgeTM for Peripheral Nerve Regeneration.

Silk fibroin (Bombyx mori) was used to manufacture a nerve conduit (SilkBridgeTM) characterized by a novel 3D architecture. The wall of the conduit consists of two electrospun layers (inner and outer) and one textile layer (middle), perfectly integrated at the structural and functional level. The manufacturing technology conferred high compression strength on the device, thus meeting clinical requirements for physiological and pathological compressive stresses. As demonstrated in a previous work, the silk material has proven to be able to provide a valid substrate for cells to grow on, differentiate and start the fundamental cellular regenerative activities in vitro and, in vivo, at the short time point of 2 weeks, to allow the starting of regenerative processes in terms of good integration with the surrounding tissues and colonization of the wall layers and of the lumen with several cell types. In the present study, a 10 mm long gap in the median nerve was repaired with 12 mm SilkBridgeTM conduit and evaluated at middle (4 weeks) and at longer time points (12 and 24 weeks). The SilkBridgeTM conduit led to a very good functional and morphological recovery of the median nerve, similar to that observed with the reference autograft nerve reconstruction procedure. Taken together, all these results demonstrated that SilkBridgeTM has an optimized balance of biomechanical and biological properties, which allowed proceeding with a first-in-human clinical study aimed at evaluating safety and effectiveness of using the device for the reconstruction of digital nerve defects in humans.

Characterization of Physical, Mechanical, and Biological Properties of SilkBridge Nerve Conduit after Enzymatic Hydrolysis.

The in vitro degradation profile and the cytotoxicity of the degradation products of a silk fibroin (SF)-based nerve conduit (SilkBridge), with a complex three-layered wall architecture comprising both native and regenerated (electrospun) fibers, are reported. The bacterial protease type XIV from Streptomyces griseus was used as a hydrolytic agent at three different enzyme/substrate ratios (1:8, 1:80, and 1:800 w/w) to account for the different susceptibility to degradation of the native and regenerated components. The incubation time was extended up to 91 days. At fixed time points, the remaining device, the insoluble debris, and the incubation buffers containing soluble degradation products were separated and analyzed. The electrospun fibers forming the inner and outer layers of the conduit wall were almost completely degraded within 10 days of incubation at an enzyme/substrate ratio of 1:80 w/w. The progression of degradation was highlighted by the emergence of zones of erosion and discontinuity along the electrospun fibers, weakening of the electrospun layers, and decrease in resistance to compressive stress. Native SF microfibers forming the middle layer of the conduit wall displayed a higher resistance to enzymatic degradation. When incubated at an enzyme/substrate ratio of 1:8 w/w, the weight decreased gradually over the incubation time as a consequence of fiber erosion and fragmentation. Analogously, the tensile properties markedly decreased. Both spectroscopic and thermal analyses confirmed the gradual increase in the crystalline character of the fibers. The incubation buffers containing the soluble degradation products were subjected to cytotoxicity testing with human HEK293 cells and mouse neuroblastoma N2a cells. No detrimental effects on cell viability were observed, suggesting that the degradation products do not retain any toxic property. Finally, the mass spectrometry analysis of degradation products showed that the SF polypeptides recovered in the incubation buffers were representative of the aminoacidic sequence of the fibroin light chain and of the highly repetitive fibroin heavy chain, indicating that virtually the entire sequence of the fibroin protein constituent of SilkBridge was degraded.

Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests.

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.

Influence of ex vivo perfusion on the biomolecular profile of rat lungs.

Despite increasing clinical adoption, biologic influences of ex vivo lung perfusion (EVLP) remain insufficiently elucidated. The aim of the current study was to investigate biomolecular changes induced by EVLP in rat lungs. EVLP was maintained for 180 min. Hyaluronan, mediators, and cells were assessed in the perfusate. Gene expression, signaling pathways, and ATP content were investigated in lung tissue. EVLP induced the release of medium-high molecular weight hyaluronan and transcription of hyaluronan synthases ( P < 0.001). Increasing concentrations of inflammatory mediators were detected in the perfusate ( P < 0.001). Perfused lungs exhibited a distinctive transcriptional signature compared with organs examined before or after surgery/procurement ( P = 0.003). Up-regulated genes were involved in inflammation and its regulation, apoptosis/survival, heat shock, and oxidative stress response ( q = 0). Down-regulated genes were related to lymphocyte function ( q = 0). The NF-κB, signal transducer and activator of transcription 3, ERK1/2, p38, Akt, and stress-activated protein kinase/JNK signaling pathways were modulated by EVLP ( P < 0.05). Most of these biomolecular changes were examined and confirmed in additional experiments that were performed in lungs procured from donation after cardiocirculatory death after 180 min of warm ischemia. The current study demonstrates that EVLP broadly affects the lung biomolecular phenotype. These findings improve our comprehension of the effects exerted by the procedure and encourage additional research in preclinical models to implement therapeutic interventions.-Lonati, C., Bassani, G. A., Brambilla, D., Leonardi, P., Carlin, A., Faversani, A., Gatti, S., Valenza, F. Influence of ex vivo perfusion on the biomolecular profile of rat lungs.