Portable NIR spectrometer for quick identification of fat bloom in chocolates.

Cocoa butter provides desirable sensory properties to chocolates; however, the exposure of chocolate to temperature variations during transportation and/or storage can lead to changes in the polymorphic form of butter, with the appearance of a dull-white film on the chocolate surface, known as fat bloom. This study investigated the use of a portable NIR spectrometer combined with chemometric tools to discriminate milk chocolate, white chocolate, 40% cocoa chocolate, and 70% cocoa chocolate samples, which were subjected to temperature abuse for 6 hours. The PCA allowed separating the samples into three classes: control at 20 °C, chocolate subjected to 35 °C, and chocolate subjected to 40 °C, for each type of chocolate studied. The PLS-DA models provided sensibility, specificity, and accuracy values in the range of 80 to 100%, and allowed identifying the wavelengths associated with the different chocolates that most impacted the construction of the models.

Tuning the matrix metalloproteinase-1 degradability of peptide amphiphile nanofibers through supramolecular engineering.

Matrix metalloproteinases (MMPs) are a family of endopeptidases capable of degrading extracellular matrix (ECM) components. They are known to play crucial roles during the ECM turnover in both physiological and pathological processes. As such, their activities are utilized as biological stimuli to engineer MMP-responsive peptide-based biomaterials such as self-assembled peptide amphiphiles (PAs). Although previous studies have unveiled the role of PAs secondary structure on the mechanical and biological properties of their self-assembled nanostructures, the effect on the degradability of their assemblies by MMP-1 has not been reported. Herein, a series of PAs are designed and synthesized, all comprising the same MMP-1 cleavable domain but with variable structural segments, to decipher the role of PA's secondary structure on the MMP-1 degradability of their assemblies. This study reveals a correlation between the MMP-1 degradation efficiency and the ß-sheet content of the self-assembled PA nanofibers, with the MMP-1 cleavability being significantly reduced in the PA nanofibers with stronger ß-sheet characteristics. These results shed light on the role of supramolecular cohesion in PA assemblies on their hydrolysis by MMP-1 and open up the possibility to control the degradation rate of PA-based nanostructures by MMP-1 through tweaking their molecular sequences.

Co-assembly, spatiotemporal control and morphogenesis of a hybrid protein-peptide system.

Controlling molecular interactions between bioinspired molecules can enable the development of new materials with higher complexity and innovative properties. Here we report on a dynamic system that emerges from the conformational modification of an elastin-like protein by peptide amphiphiles and with the capacity to access, and be maintained in, non-equilibrium for substantial periods of time. The system enables the formation of a robust membrane that displays controlled assembly and disassembly capabilities, adhesion and sealing to surfaces, self-healing and the capability to undergo morphogenesis into tubular structures with high spatiotemporal control. We use advanced microscopy along with turbidity and spectroscopic measurements to investigate the mechanism of assembly and its relation to the distinctive membrane architecture and the resulting dynamic properties. Using cell-culture experiments with endothelial and adipose-derived stem cells, we demonstrate the potential of this system to generate complex bioactive scaffolds for applications such as tissue engineering.

Molecularly engineered self-assembling membranes for cell-mediated degradation.

The use of peptide engineering to develop self-assembling membranes that are responsive to cellular enzyme activities is reported. The membranes are obtained by combining hyaluronan (HA) and a rationally designed peptide amphiphile (PA) containing a proteolytic domain (GPQGIWGQ octapeptide) sensitive to matrix metalloproteinase-1 (MMP-1). Insertion of an octapeptide in a typical PA structure does not disturb its self-assembly into fibrillar nanostructures neither the ability to form membranes with HA. In vitro enzymatic degradation with hyaluronidase and MMP-1 shows that membranes containing the MMP-1 substrate exhibit enhanced enzymatic degradation, compared with control membranes (absence of MMP-1 cleavable peptide or containing a MMP-1 insensitive sequence), being completely degraded after 7 days. Cell viability and proliferation is minimally affected by the enzymatically cleavable functionality of the membrane, but the presence of MMP-1 cleavable sequence does stimulate the secretion of MMP-1 by fibroblasts and interfere with matrix deposition, particularly the deposition of collagen. By showing cell-responsiveness to biochemical signals presented on self-assembling membranes, this study highlights the ability of modulating certain cellular activities through matrix engineering. This concept can be further explored to understand the cellular remodeling process and as a strategy to develop artificial matrices with more biomimetic degradation for tissue engineering applications.

Hyaluronan and self-assembling peptides as building blocks to reconstruct the extracellular environment in skin tissue.

Self-assembling bioactive membranes, incorporating hyaluronan, a structural component of the skin extracellular matrix (ECM), and peptide amphiphiles presenting biochemical signals, are proposed in this work for recapitulating some aspects of the skin tissue microenvironment. In the herein presented strategy, the availability of cell-adhesion ligands (0-50% RGDS epitope) within 2D membranes is controlled aiming at mastering the adhesion of human dermal fibroblasts under serum-free culture conditions. The membranes were characterized with respect to their microstructure, by scanning electron microscopy (SEM), epitope distribution, degradability and cell behavior, regarding adhesion, proliferation and cytoskeleton organization. SEM of the membrane surface showed a network of nanofibers that are remarkably reminiscent of the filamentous structure found in the ECM. Confocal microscopy images, using a fluorescently labeled RGDS-peptide, showed that the RGDS signal is uniformly distributed on the membranes. Degradation studies indicated that the membranes are susceptible to enzymatic degradation by hyaluronidase. In the presence of the enzyme at physiological concentration, the membranes degrade gradually over time. When grown on membranes with the cell recognition epitope RGDS, fibroblasts had spread out and elongated, exhibiting extended filopodia interacting with fibrillar structure of the membrane surface, thus showing improved adhesion to the substrate. This study demonstrates the positive effect of the RGDS epitope, presented on a self-assembled membrane, in promoting cell-matrix interactions.

Alginate encapsulation as a novel strategy for the cryopreservation of neurospheres.

Primary cultures of brain cell neurospheres are valuable in vitro models for neurotoxicology and brain cell research. Such applications would greatly benefit from the development of efficient cryopreservation protocols that assure the availability of viable and genetically stable stocks of functional neurospheres. In this work we aimed at developing an integrated strategy allowing for long-term culture and cryopreservation of brain cell neurospheres with high viability and reduced recovery time postthawing. Microencapsulation in clinical-grade, ultrahigh viscous, highly purified alginate uniformly cross-linked with Ba(2+) was evaluated as the main strategy to avoid the commonly observed loss of cell-cell and cell-matrix interactions with consequent aggregate's fragmentation and decrease in cell viability that occurs postthawing. Brain cells isolated from 16-day-old fetal rats were cultured in spinner vessels as neurospheres, encapsulated at the 5th day of culture, and cryopreserved at day 19. Culture characterization and assessment of postthawing recovery, concerning cell metabolism, aggregate's cell type composition, and neuron-astrocyte interactions were performed through analysis of membrane integrity, metabolic activity assays, and immunohistochemistry. Our results show that the encapsulation process does not affect cell viability's central metabolism; neither cell differentiation nor cell extensions into cell networks are usually observed between neurons and astrocytes within the neurosphere structure. Neurosphere encapsulation resulted in reduced recovery time postthawing and significantly less fragmentation. Further, the use of serum-free CryoStor™ solution provided further protection for both nonencapsulated and encapsulated aggregates compared with serum-supplemented culture medium as the cryopreservation medium.