Isolation and Characterization of Cellulose Microfibers from Colombian Cocoa Pod Husk via Chemical Treatment with Pressure Effects.

One of the current challenges is to add value to agro-industrial wastes, and the cocoa industry generates about 10 tons of cocoa pod husks in Colombia for each ton of cocoa beans, which are incinerated and cause environmental damage. This study characterized the Colombian cocoa pod husk (CPH) and to isolate and characterize cellulose microfibers (tCPH) extracted via chemical treatment and pressure. Chemical and physical analyses of CPH were performed, and a pretreatment method for CPH fibers was developed, which is followed by a hydrolysis method involving high pressure in an autoclave machine with an alkaline medium (6% NaOH), and finally, bleaching of the fiber to obtain tCPH. The tCPH cellulose microfibers were also chemically and physically analyzed and characterized by infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and thermo-gravimetric analysis (TGA). Chemical and physical characterization showed a decrease in lignin content in tCPH. FTIR analysis showed the absence of some peaks in tCPH with respect to the CPH spectrum; XRD results showed an increase in crystallinity for tCPH compared to CPH, due to a higher presence of crystalline cellulose in tCPH. SEM images included a control fiber treated without high pressure (tCPHnpe), and agglomerated fibers were observed, whereas cellulose microfibers with a mean diameter of 10 ± 2.742 µm were observed in tCPH. Finally, with TGA and DTGA it was confirmed that in tCPH, the hemicellulose and lignin were removed more successfully than in the control fiber (tCPHnpe), showing that the treatment with pressure was effective at isolating the cellulose microfibers from cocoa pod husk.

Assessing cellular internalization and endosomal escape abilities of novel BUFII-Graphene oxide nanobioconjugates.

Cell-penetrating agents based on functionalized nanoplatforms have emerged as a promising approach for developing more efficient and multifunctional delivery vehicles for treating various complex diseases that require reaching different intracellular compartments. Our previous work has shown that achieving full cellular coverage and high endosomal escape rates is possible by interfacing magnetite nanoparticles with potent translocating peptides such as Buforin II (BUF-II). In this work, we extended such an approach to two graphene oxide (GO)-based nanoplatforms functionalized with different surface chemistries to which the peptide molecules were successfully conjugated. The developed nanobioconjugates were characterized via spectroscopic (FTIR, Raman), thermogravimetric, and microscopic (SEM, TEM, and AFM) techniques. Moreover, biocompatibility was assessed via standardized hemocompatibility and cytotoxicity assays in two cell lines. Finally, cell internalization and coverage and endosomal escape abilities were estimated with the aid of confocal microscopy analysis of colocalization of the nanobioconjugates with Lysotracker Green®. Our findings showed coverage values that approached 100% for both cell lines, high biocompatibility, and endosomal escape levels ranging from 30 to 45% and 12-24% for Vero and THP-1 cell lines. This work provides the first routes toward developing the next-generation, carbon-based, cell-penetrating nanovehicles to deliver therapeutic agents. Further studies will be focused on elucidating the intracellular trafficking pathways of the nanobioconjugates to reach different cellular compartments.

Blood-Vessel-Inspired Hierarchical Trilayer Scaffolds: PCL/Gelatin-Driven Protein Adsorption and Cellular Interaction.

Fabrication of scaffolds with hierarchical structures exhibiting the blood vessel topological and biochemical features of the native extracellular matrix that maintain long-term patency remains a major challenge. Within this context, scaffold assembly using biodegradable synthetic polymers (BSPs) via electrospinning had led to soft-tissue-resembling microstructures that allow cell infiltration. However, BSPs fail to exhibit the sufficient surface reactivity, limiting protein adsorption and/or cell adhesion and jeopardizing the overall graft performance. Here, we present a methodology for the fabrication of three-layered polycaprolactone (PCL)-based tubular structures with biochemical cues to improve protein adsorption and cell adhesion. For this purpose, PCL was backbone-oxidized (O-PCL) and cast over a photolithography-manufactured microgrooved mold to obtain a bioactive surface as demonstrated using a protein adsorption assay (BSA), Fourier transform infrared spectroscopy (FTIR) and calorimetric analyses. Then, two layers of PCL:gelatin (75:25 and 95:5 w/w), obtained using a novel single-desolvation method, were electrospun over the casted O-PCL to mimic a vascular wall with a physicochemical gradient to guide cell adhesion. Furthermore, tensile properties were shown to withstand the physiological mechanical stresses and strains. In vitro characterization, using L929 mouse fibroblasts, demonstrated that the multilayered scaffold is a suitable platform for cell infiltration and proliferation from the innermost to the outermost layer as is needed for vascular wall regeneration. Our work holds promise as a strategy for the low-cost manufacture of next-generation polymer-based hierarchical scaffolds with high bioactivity and resemblance of ECM's microstructure to accurately guide cell attachment and proliferation.

Failure Analysis of TEVG's II: Late Failure and Entering the Regeneration Pathway.

Tissue-engineered vascular grafts (TEVGs) are a promising alternative to treat vascular disease under complex hemodynamic conditions. However, despite efforts from the tissue engineering and regenerative medicine fields, the interactions between the material and the biological and hemodynamic environment are still to be understood, and optimization of the rational design of vascular grafts is an open challenge. This is of special importance as TEVGs not only have to overcome the surgical requirements upon implantation, they also need to withhold the inflammatory response and sustain remodeling of the tissue. This work aims to analyze and evaluate the bio-molecular interactions and hemodynamic phenomena between blood components, cells and materials that have been reported to be related to the failure of the TEVGs during the regeneration process once the initial stages of preimplantation have been resolved, in order to tailor and refine the needed criteria for the optimal design of TEVGs.

Computational Characterization of Mechanical, Hemodynamic, and Surface Interaction Conditions: Role of Protein Adsorption on the Regenerative Response of TEVGs.

Currently available small diameter vascular grafts (<6 mm) present several long-term limitations, which has prevented their full clinical implementation. Computational modeling and simulation emerge as tools to study and optimize the rational design of small diameter tissue engineered vascular grafts (TEVG). This study aims to model the correlation between mechanical-hemodynamic-biochemical variables on protein adsorption over TEVG and their regenerative potential. To understand mechanical-hemodynamic variables, two-way Fluid-Structure Interaction (FSI) computational models of novel TEVGs were developed in ANSYS Fluent 2019R3® and ANSYS Transient Structural® software. Experimental pulsatile pressure was included as an UDF into the models. TEVG mechanical properties were obtained from tensile strength tests, under the ISO7198:2016, for novel TEVGs. Subsequently, a kinetic model, linked to previously obtained velocity profiles, of the protein-surface interaction between albumin and fibrinogen, and the intima layer of the TEVGs, was implemented in COMSOL Multiphysics 5.3®. TEVG wall properties appear critical to understand flow and protein adsorption under hemodynamic stimuli. In addition, the kinetic model under flow conditions revealed that size and concentration are the main parameters to trigger protein adsorption on TEVGs. The computational models provide a robust platform to study multiparametrically the performance of TEVGs in terms of protein adsorption and their regenerative potential.

Highly Efficient Synthesis of Type B Gelatin and Low Molecular Weight Chitosan Nanoparticles: Potential Applications as Bioactive Molecule Carriers and Cell-Penetrating Agents.

Gelatin and chitosan nanoparticles have been widely used in pharmaceutical, biomedical, and nanofood applications due to their high biocompatibility and biodegradability. This study proposed a highly efficient synthesis method for type B gelatin and low-molecular-weight (LMW) chitosan nanoparticles. Gelatin nanoparticles (GNPs) were synthesized by the double desolvation method and the chitosan nanoparticles (CNPs) by the ionic gelation method. The sizes of the obtained CNPs and GNPs (373 ± 71 nm and 244 ± 67 nm, respectively) and zeta potential (+36.60 ± 3.25 mV and -13.42 ± 1.16 mV, respectively) were determined via dynamic light scattering. Morphology and size were verified utilizing SEM and TEM images. Finally, their biocompatibility was tested to assure their potential applicability as bioactive molecule carriers and cell-penetrating agents.

Bioactive Poly(lactic acid)-Cocoa Bean Shell Composites for Biomaterial Formulation: Preparation and Preliminary In Vitro Characterization.

The unique lignocellulosic and solvent-extractive chemical constituents of most natural fibers are rich in natural polymers and bioactive molecules that can be exploited for biomaterial formulation. However, although natural fibers' main constituents have been already incorporated as material reinforcement and improve surface bioactivity of polymeric materials, the use of the whole natural fibers as bioactive fillers remains largely unexplored. Thus, we put forward the formulation of natural fiber filling and functionalization of biomaterials by studying the chemical composition of cocoa bean shells (CBS) and proposing the fabrication and characterization of polylactic acid (PLA) and CBS-based composite by solvent-casting. As was expected from previous studies of agro-industrial wastes, the main components of CBS were to cellulose (42.23 wt.%), lignin (22.68 wt.%), hemicellulose (14.73 wt.%), and solvent extractives (14.42 wt.%). Structural analysis (FTIR) confirms the absence of covalent bonding between materials. Thermal degradation profiles (DSC and TGA) showed similar mass losses and thermal-reaction profiles for lignocellulosic-fibers-based composites. The mechanical behavior of the PLA/CBS composite shows a stiffer material behavior than the pristine material. The cell viability of Vero cells in the presence of the composites was above 94%, and the hemolytic tendency was below 5%, while platelet aggregation increased up to 40%. Antioxidant activity was confirmed with comparable 2,2-diphe-277 nyl-1-picryl-hydrazyl-hydrate (DPPH) free-radical scavenging than Vitamin C even for PLA/CBS composite. Therefore, the present study elucidates the significant promise of CBS for bioactive functionalization in biomaterial-engineering, as the tested composite exhibited high biocompatibility and strong antioxidant activity and might induce angiogenic factors' release. Moreover, we present an eco-friendly alternative to taking advantage of chocolate-industry by-products.