Surface structure and properties of poly-(ethylene terephthalate) hydrolyzed by alkali and cutinase

This study was aimed at comparatively investigating the hydrolysis of crystalline and amorphous poly-(ethylene terephthalate) films by alkali and cutinase. Changes of surface properties were investigated by FTIR spectroscopy (ATR mode). The A1341/A1410 and I1120/I1100 absorbance ratios, and the full width at half maximum of the carbonyl stretching band (FWHM1715) were used to evaluate the polymer crystallinity and its changes upon hydrolysis. The effect of different treatments on chain orientation was evaluated by calculating R ratios of appropriate bands. The spectroscopic indexes showed that both alkali and enzyme treatments induced structural and conformational rearrangements with a consequent increase in crystallinity in both amorphous and crystalline films. The crystalline PET film was modified more strongly by alkali than by cutinase, while the opposite occurred for the amorphous one. The trend of the water contact angle (WCA) clearly indicates that alkali is more effective than cutinase in enhancing hydrophilicity of PET films and that the effect is stronger on amorphous than on crystalline films. The values of WCA correlate well with the FTIR indexes calculated from the spectra of hydrolyzed crystalline PET films. The mechanism of the surface hydrolysis of PET by alkali and cutinase is discussed

Vascular Tissue Engineering: Recent Advances in Small Diameter Blood Vessel Regeneration

Cardiovascular diseases are the leading cause of mortality around the globe. The development of a functional and appropriate substitute for small diameter blood vessel replacement is still a challenge to overcome the main drawbacks of autografts and the inadequate performances of synthetic prostheses made of polyethylene terephthalate (PET, Dacron) and expanded polytetrafluoroethylene (ePTFE, Goretex). Therefore, vascular tissue engineering has become a promising approach for small diameter blood vessel regeneration as demonstrated by the increasing interest dedicated to this field. This review is focused on the most relevant and recent studies concerning vascular tissue engineering for small diameter blood vessel applications. Specifically, the present work reviews research on the development of tissue-engineered vascular grafts made of decellularized matrices and natural and/or biodegradable synthetic polymers and their realization without scaffold

Silk Vascular Grafts with Optimized Mechanical Properties for the Repair and Regeneration of Small Caliber Blood Vessels

As the incidence of cardiovascular diseases has been growing in recent years, the need for small-diameter vascular grafts is increasing. Considering the limited success of synthetic grafts, vascular tissue engineering/repair/regeneration aim to find novel solutions. Silk fibroin (SF) has been widely investigated for the development of vascular grafts, due to its good biocompatibility, tailorable biodegradability, excellent mechanical properties, and minimal inflammatory reactions. In this study, a new generation of three-layered SF vascular scaffolds has been produced and optimized. Four designs of the SILKGraft vascular prosthesis have been developed with the aim of improving kink resistance and mechanical strength, without compromising the compliance with native vessels and the proven biocompatibility. A more compact arrangement of the textile layer allowed for the increase in the mechanical properties along the longitudinal and circumferential directions and the improvement of the compliance value, which approached that reported for the saphenous and umbilical veins. The higher braid density slightly affected the grafts' morphology, increasing surface roughness, but the novel design mimicked the corrugation approach used for synthetic grafts, causing significant improvements in kink resistance

Human Keratinocytes and Fibroblasts Co-Cultured on Silk Fibroin Scaffolds Exosomally Overrelease Angiogenic and Growth Factors

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 regen- erated 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-alpha/beta/gamma, IL-1beta, 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

In-vivo evaluation of silk fibroin small-diameter vascular grafts: state of art of preclinical studies and animal models

Autologous vein and artery remains the first choice for vascular grafting procedures in small-diameter vessels such as coronary and lower limb districts. Unfortunately, these vessels are often found to be unsuitable in atherosclerotic patients due to the presence of calcifications or to insufficient size. Synthetic grafts composed of materials such as expanded polytetrafluoroethylene (ePTFE) are frequently employed as second choice, because of their widespread availability and success in the reconstruction of larger arteries. However, ePTFE grafts with small diameter are plagued by poor patency rates due to surface thrombogenicity and intimal hyperplasia, caused by the bioinertness of the synthetic material and aggravated by low flow conditions. Several bioresorbable and biodegradable polymers have been developed and tested to exploit such issues for their potential stimulation to endothelialization and cell infiltration. Among these, silk fibroin (SF) has shown promising pre-clinical results as material for small-diameter vascular grafts (SDVGs) because of its favorable mechanical and biological properties. A putative advantage in graft infection in comparison with synthetic materials is plausible, although it remains to be demonstrated. Our literature review will focus on the performance of SF-SDVGs in vivo, as evaluated by studies performing vascular anastomosis and interposition procedures, within small and large animal models and different arterial districts. Efficiency under conditions that more accurately mime the human body will provide encouraging evidence towards future clinical application

Exosomes of adult human fibroblasts cultured on 3D silk fibroin nonwovens intensely stimulate neoangiogenesis

Bombyx mori silk fibroin (SF) is a biomacromolecule allowing to assemble scaffolds 7 for tissue engineering/regeneration purposes because of its cellular adhesiveness, high 8 biocompatibility, and low immunogenicity. Earlier work showed that two types of 3D-SF-based 9 nonwovens (3D-SFnws) implanted into mouse subcutaneous tissue were promptly vascularised via plates in exosome-depleted medium. DNA amounts and D-glucose consumption revealed HDFs 17 undefined molecular mechanisms. This study used nontumorigenic adult human dermal fibroblasts (HDFs) adhering to a third type of 3D-SFnws to test whether HDFs release exosomes whose contents promote neoangiogenesis

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