The degeneration of photoreceptors in the retina is one of the major causes of adult blindness in humans. Unfortunately, no effective clinical treatments exist for the majority of retinal ...degenerative disorders. Here we report on the fabrication and functional validation of a fully organic prosthesis for long-term in vivo subretinal implantation in the eye of Royal College of Surgeons rats, a widely recognized model of retinitis pigmentosa. Electrophysiological and behavioural analyses reveal a prosthesis-dependent recovery of light sensitivity and visual acuity that persists up to 6-10 months after surgery. The rescue of the visual function is accompanied by an increase in the basal metabolic activity of the primary visual cortex, as demonstrated by positron emission tomography imaging. Our results highlight the possibility of developing a new generation of fully organic, highly biocompatible and functionally autonomous photovoltaic prostheses for subretinal implants to treat degenerative blindness.
In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were ...successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their k cat and K m values on pNP–acetate were in the ranges 2.4–211.9 s–1 and 127–200 μM while on pNP–butyrate they showed k cat and K m values between 5.3 and 195.1 s–1 and between 1483 and 2133 μM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A 1340/A 1410 upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the O of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.
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
A
1341/
A
1410 and
I
1120/
I
1100 absorbance ratios, and the full width at half maximum of the carbonyl stretching band (FWHM
1715) 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.
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.
The capability of
Agaricus bisporus tyrosinase to catalyze the oxidation of tyrosine residues of silk sericin was studied under homogeneous reaction conditions, by using sericin peptides purified ...from industrial wastewater as the substrate. Tyrosinase was able to oxidize about 57% of sericin-bound tyrosine residues. The reaction rate was higher than with silk fibroin, but lower than with other silk-derived model peptides, i.e. tryptic and chymotryptic soluble peptide fractions of silk fibroin, suggesting that the size and the molecular conformation of the substrate influenced the kinetics of the reaction. The concentration of tyrosine in oxidized sericin samples decreased gradually with increasing the enzyme-to-substrate ratio. The average molecular weight of sericin peptides significantly increased by oxidation, indicating that cross-linking occurred via self-condensation of
o-quinones and/or coupling with the free amine groups of lysine and, probably, with sulfhydryl groups of cysteine. The high temperature shift of the main thermal transitions observed in the differential scanning calorimetry curves confirmed the formation of peptide species with higher molecular weight and higher thermal stability. Fourier transform-infrared spectra of oxidized sericin samples showed slight changes related to the loss of tyrosine and formation of oxidation products. Oxidized sericin peptides were able to undergo non-enzymatic coupling with chitosan. Infrared spectra provided clear evidence of the formation of sericin–chitosan bioconjugates under homogeneous reaction conditions. Spectral changes in the NH stretching region seem to support the formation of bioconjugates via the Michael addition mechanism.
The domesticated silkworm,
Bombyx mori
, is a fundamental insect for silk industry. Silk is obtained from cocoons, protective envelopes produced during pupation and composed of single raw silk ...filaments secreted by the insect silk glands. Currently, silk is used as a textile fibre and to produce new materials for technical and biomedical applications. To enhance the use of both fabrics and silk-based materials, great efforts to obtain silk with antimicrobial properties have been made. In particular, a convincing approach is represented by the enrichment of the textile fibre with antimicrobial peptides, the main effectors of the innate immunity. To this aim, silkworm-based transgenic techniques appear to be cost-effective strategies to obtain cocoons in which antimicrobial peptides are integrated among the silk proteins. Recently, cocoons transgenic for a recombinant silk protein conjugated to the silkworm Cecropin B antimicrobial peptide were obtained and showed enhanced antibacterial properties (Li et al. in Mol Biol Rep 42:19–25,
https://doi.org/10.1007/s11033-014-3735-z
,
2015a
). In this work we used the
piggyBac
-mediated germline transformation to generate several transgenic
B. mori
lines able to overexpress Cecropin B or Moricin antimicrobial peptides at the level of the silk gland. The derived cocoons were characterised by increased antimicrobial properties and the resulting silk fibre was able to inhibit the bacterial growth of the Gram-negative
Escherichia coli.
Our results suggest that the generation of silkworm overexpressing unconjugated antimicrobial peptides in the silk gland might represent an additional strategy to obtain antimicrobial peptide-enriched silk, for the production of new silk-based materials.
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.
Consistent with this endeavor, we used regenerated silk fibroin (SF) from
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.
: 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 (
) 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
) significantly more strongly stimulated human dermal microvascular endothelial cells to migrate and assemble tubes/nodes in vitro.
: 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.
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