Fabrication of 3D printed antimicrobial polycaprolactone scaffolds for tissue engineering applications.

Synthetic polymers are widely employed for bone tissue engineering due to their tunable physical properties and biocompatibility. Inherently, most of these polymers display poor antimicrobial properties. Infection at the site of implantation is a major cause for failure or delay in bone healing process and the development of antimicrobial polymers is highly desired. In this study, silver nanoparticles (AgNps) were synthesized in polycaprolactone (PCL) solution by in-situ reduction and further extruded into PCL/AgNps filaments. Customized 3D structures were fabricated using the PCL/AgNps filaments through 3D printing technique. As demonstrated by scanning electron microscopy, the 3D printed scaffolds exhibited interconnected porous structures. Furthermore, X-ray photoelectron spectroscopy analysis revealed the reduction of silver ions. Transmission electron microscopy along with energy-dispersive X-ray spectroscopy analysis confirmed the formation of silver nanoparticles throughout the PCL matrix. In vitro enzymatic degradation studies showed that the PCL/AgNps scaffolds displayed 80% degradation in 20 days. The scaffolds were cytocompatible, as assessed using hFOB cells and their antibacterial activity was demonstrated on Escherichia coli. Due to their interconnected porous structure, mechanical and antibacterial properties, these cytocompatible multifunctional 3D printed PCL/AgNps scaffolds appear highly suitable for bone tissue engineering.

Morphological and elemental mapping of gallstones using synchrotron microtomography and synchrotron X-ray fluorescence spectroscopy.

BACKGROUND AND AIM: Regional differences in gallstone (GS) composition are well documented in the Indian subcontinent. The reasons for the same are unknown. Etiopathogenesis of GS remains elusive despite advances in instrumentation. This was an in-depth analysis of the chemical, structural, and elemental composition of GS with special reference to synchroton studies. METHODS: We used high-end sensitive analytical complementary microscopic and spectroscopic methods techniques, such as X-ray diffraction, scanning electron microscopy, Fourier transform infrared, synchrotron X-ray fluorescence spectroscopy (SR-XRF), and 2D and 3D synchrotron microtomography (SR-µCT), to study the ultra structure and trace element composition of three major types of GS (cholesterol, mixed, and pigment). SR-XRF quantified the trace elements in GS. RESULTS: The cholesterol GS (monohydrate and anhydrate) were crystalline, with high calcium content. The pigment GS were amorphous, featureless, black, and fragile, with high calcium bilirubinate and carbonate salts. They had the highest concentration of iron (average 31.50 ppm) and copper (average 92.73 ppm), with bacterial inclusion. The mixed stones had features of both cholesterol and pigment GS with intermediate levels of copper (average 20.8 ppm) and iron (average 17.78 ppm). CONCLUSION: SR-µCT has, for the first time, provided cross-sectional computed imaging delineating the framework of GS and mineral distribution. It provided excellent mapping of cholesterol GS. SR-XRF confirmed that pigment GS had high concentrations of copper and iron with bacterial inclusions, the latter possibly serving as a nidus to the formation of these stones.

Effect of passaging on the stemness of infrapatellar fat pad­derived stem cells and potential role of nucleostemin as a prognostic marker of impaired stemness.

Infrapatellar fat pad­derived stem cells (IFPSCs) are emerging as an alternative to adipose tissue­derived stem cells (ADSCs) from other sources. They are a reliable source of autologous stem cells obtained from medical waste that are suitable for use in cell­based therapy, tissue engineering and regenerative medicine. Such clinical applications require a vast number of high­quality IFPSCs. Unlike embryonic stem cells (ESCs), ADSCs and IFPSCs have limited population doubling capacity; however, in vitro expansion of primary IFPSCs through multiple passages (referred to as P) is a crucial step to acquire the desired population of cells. The present study investigated the effect of multiple passages on the stemness of IFPSCs during expansion and the possibility of predicting the loss of stemness using certain markers. IFPSCs were isolated from infrapatellar fat pad tissue resected during knee arthroplasty performed on aged patients (>65 years old). These cells from the stromal vascular fraction were serially passaged to at least to P7, and their stemness characteristics were examined at each passage. It was observed that IFPSCs maintained their spindle­shaped morphology, self­renewability and homogeneity at P2­4. Furthermore, immunostaining revealed that these cells expressed mesenchymal stem cell (CD166, CD90 and CD105) and ESC markers [Sox2, Nanog, Oct4 and nucleostemin (NS)], whereas the hematopoietic stem cell marker CD45 was absent. These cells were also able to differentiate into the three germ layer cell types, thus confirming their ability to generate clinical grade cells. The findings indicated that prolonged culture of IFPSCs (P>6) led to the loss of the stem cell proliferative marker NS, with an increased population doubling time and progression toward neuronal differentiation, acquiring a neurogenic phenotype. Additionally, IFPSCs demonstrated an inherent ability to secrete neurotrophic factors and express receptors for these factors, which is the cause of neuronal differentiation at later passages. Therefore, these findings validated NS as a prognostic indicator for impaired stemness and identified IFPSCs as a promising source for cell­based therapy, particularly for neurodegenerative diseases.

Porous Gelatin Membrane Obtained from Pickering Emulsions Stabilized by Graphene Oxide.

This article presents a novel procedure for preparing porous membranes from water-soluble polymers involving the formation of a Pickering emulsion. Gelatin is a biodegradable biopolymer obtained by the partial hydrolysis of collagen. A biopolymer such as gelatin is capable of adsorbing at an oil/water interface, resulting in decreased interfacial energy. Hence, gelatin is widely employed as an alternate for synthetic surfactants to stabilize emulsions in the food industry. However, high-molecular-weight gelatin leads to large emulsion droplets and poor emulsion stability. The amphoteric nature of graphene oxide (GO) nanosheets was helpful in stabilizing the oil/water interface and allows for the preparation of a stable gelatin/GO emulsion. Membranes fabricated using gelatin/GO have a uniformly distributed porous structure. However, prepared membranes are highly hydrosoluble, so the membranes were cross-linked without affecting their morphology. XRD results evidenced that gelatin effectively exfoliated the graphite oxide which is essential to stabilizing the emulsion. Fabricated gelatin/GO membranes possess uniformly distributed pores and are highly stable in aqueous solution. Pure water filtration tests were conducted on the membranes. The permeability results proved that the membranes fabricated by a Pickering emulsion are promising materials for filtration.

Design of Boron Nitride/Gelatin Electrospun Nanofibers for Bone Tissue Engineering.

Gelatin is a biodegradable biopolymer obtained by collagen denaturation, which shows poor mechanical properties. Hence, improving its mechanical properties is very essential toward the fabrication of efficient nontoxic material for biomedical applications. For this aim, various methods are employed using external fillers such as ceramics or bioglass. In this report, we introduce boron nitride (BN)-reinforced gelatin as a new class of two-dimensional biocompatible nanomaterials. The effect of the nanofiller on the mechanical behavior is analyzed. BN is efficiently exfoliated using the biopolymer gelatin as shown through Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD). The exfoliated BN reinforces gelatin electrospun fibers, which results in an increase in the Young's modulus. The Electrospun Mats (ESM) are stable after the glutaraldehyde cross-linking, and the fibrous morphology is preserved. The cross-linked gelatin/BN ESM is highly bioactive in forming bonelike hydroxyapatite as shown by scanning electron microscopy. Due to their enhanced mineralization ability, the cross-linked ESM have been tested on human bone cells (HOS osteosarcoma cell line). The cell attachment, proliferation, and biocompatibility results show that the ESM are nontoxic and biodegradable. The analysis of osteoblast gene expression and the measurement of alkaline phosphatase activity confirm that these materials are suitable for bone tissue engineering.

Novel biocompatible electrospun gelatin fiber mats with antibiotic drug delivery properties.

The aim of this study was to synthesize stable gelatin electrospun mats (ESMs) (cross-linked by glutaraldehyde (GTA) vapors) with tunable drug release properties using pH as a stimulus. Gelatin ESMs loaded with rhodamine as a model drug were first synthesized. The in vitro release of rhodamine was characterized to understand the mechanisms of drug release and the effects of both cross-linker concentration and pH on drug release. An optimal cross-linker concentration of 5% was evidenced to provide ESMs allowing both sustainable release of drugs at pH 7 and burst release at pH 2. The release profiles were then fitted with a power law model to describe the release kinetics. The chlorhexidine antibiotic drug was finally loaded into the optimal electrospun mat and its bactericidal activity was demonstrated against Gram-negative (E. coli) and Gram-positive (S. epidermidis) bacteria by agar diffusion tests. This biocompatible material was shown to efficiently destroy bacterial biofilms and prevent bacterial growth within a short time (3 h), while maintaining its antibacterial activity up to at least 72 h. This study provides a promising material, which could treat infected sites and prevent infections, with tunable drug releasing properties using pH as a stimulus.

Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice.

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.

Structure of the heterodimeric neurotoxic complex viperotoxin F (RV-4/RV-7) from the venom of Vipera russelli formosensis at 1.9 A resolution.

The presynaptic viperotoxin F is the major lethal component of the venom of Vipera russelli formosensis (Taiwan viper). It is a heterodimer of two highly homologous (65% identity) but oppositely charged subunits: a basic and neurotoxic PLA(2) (RV-4) and an acidic non-toxic component with a very low enzymatic activity (RV-7). The crystal structure of the complex has been determined by molecular replacement and refined to 1.9 A resolution and an R factor of 22.3% with four RV-4/RV-7 complexes in the asymmetric unit, which do not exhibit any local point-group symmetry. The complex formation decreases the accessible surface area of the two subunits by approximately 1425 A(2). Both PLA(2)s are predicted to have very low, if any, anticoagulant activity. The structure of viperotoxin F is compared with that of the heterodimeric neurotoxin vipoxin from the venom of another viper, V. ammodytes meridionalis. The structural basis for the differences between the pharmacological activities of the two toxins is discussed. The neutralization of the negative charge of the major ligand for Ca(2+), Asp49, by intersubunit salt bridges is probably a common mechanism of self-stabilization of heterodimeric Viperinae snake-venom neurotoxins in the absence of bound calcium.

Enzymatic activity and inhibition of the neurotoxic complex vipoxin from the venom of Vipera ammodytes meridionalis.

Vipoxin from the venom of Vipera ammodytes meridionalis is an unique neurotoxic complex between a toxic phospholipase A2 and a highly homologous non-toxic protein inhibitor. It is an example of evolution of a catalytic and toxic function into inhibitory and non-toxic one. The activity of the V. ammodytes meridionalis toxin is 1.7 times higher than that of the closely related (92% sequence identity) neurotoxic complex RV4/RV7 from the venom of Vipera russelli formosensis The enhanced enzymatic activity of vipoxin is attributed to limited structural changes, in particular to the substitutions G54R and Q78K in the PLA2 subunit of the complex and to the T54R substitution in the inhibitor. Oleyloxyethylphosphocholine, aristolochic acid and vitamin E suppressed the enzymatic activity of vipoxin and its isolated PLA2 subunit. These compounds influence inflammatory processes in which PLA2 is implicated. The peptide Lys-Ala-Ile-Tyr-Ser, which is an integral part of the PLA2 components of the two neurotoxic complexes from V. ammodytes meridionalis and V. russelli formosensis (sequence 70-74) activated vipoxin increasing its PLA2 activity by 23%. This is in contrast to the inhibitory effect of the respective pentapeptides with 70-74 sequences on other group II PLA2s. Surprisingly, the same peptide inhibited 46% of the V. russelli formosensis PLA2 activity. The limited changes in the structure of the two highly homologous neurotoxins lead to considerable differences in their interaction with native peptides.