Kinetic Study of the Esterase-like Activity of Albumin following Condensation by Macromolecular Crowding.

The ability of bovine serum albumin (BSA) to form condensates in crowded environments has been discovered only recently. Effects of this condensed state on the secondary structure of the protein have already been unraveled as some aging aspects, but the pseudo-enzymatic behavior of condensed BSA has never been reported yet. This article investigates the kinetic profile of para-nitrophenol acetate hydrolysis by BSA in its condensed state with poly(ethylene) glycol (PEG) as the crowding agent. Furthermore, the initial BSA concentration was varied between 0.25 and 1 mM which allowed us to modify the size distribution, the volume fraction, and the partition coefficient (varying from 136 to 180). Hence, the amount of BSA originally added was a simple way to modulate the size and density of the condensates. Compared with dilute BSA, the initial velocity (vi) with condensates was dramatically reduced. From the Michaelis-Menten fits, the extracted Michaelis constant Km and the maximum velocity Vmax decreased in control samples without condensates when the BSA concentration increased, which was attributed to BSA self-oligomerization. In samples containing condensates, the observed vi was interpreted as an effect of diluted BSA remaining in the supernatants and from the condensates. In supernatants, the crowding effect of PEG increased the kcat and catalytic efficiency. Last, Vmax was proportional to the volume fraction of the condensates, which could be controlled by varying its initial concentration. Hence, the major significance of this article is the control of the size and volume fraction of albumin condensates, along with their kinetic profile using liquid-liquid phase separation.

Active Osteoblasts or Quiescent Bone Lining Cells? Preosteoblasts Fate Orchestrated by Curvature and Stiffness of an In Vitro 2.5D Biomimetic Culture System.

Biomimetic cell culture systems are required to provide more physiologically relevant microenvironments for bone cells. Here, a simple 2.5D culture platform is proposed, combining adjustable stiffness and surface features that mimic bone topography by using sandpaper grits as master molds with two stiffness formulations of polydimethylsiloxane (PDMS). The subsequent replicas perfectly conform the grits and reproduce the corresponding negative relief with cavities separated by convex edges. Biomimicry is also provided by an extracellular matrix (ECM)-like thin film coating, using the layer-by-layer (LbL) method. The topographical features, alternating concave, and convex structures drive preosteoblasts organization and morphology. Strikingly, curvature orchestrates the commitment of preosteoblasts, with i) maturation to active osteoblasts able to produce a dense collagenous matrix that ultimately mineralizes in the cavities, and ii) edges hosting quiescent cells that synthetize a very thin immature collagen layer with no mineralization. In summary, the present in vitro culture system model offers a cell-instructive 2.5D microenvironment that controls preosteoblasts fate, leading to two coexisting subpopulations: mature osteoblasts and bone lining cells (BLC). This promising culture system opens new avenues to advanced tissue-engineered modeling and can be applied to precellularized bone biomaterials.

Dual-functional antibacterial and osteogenic nisin-based layer-by-layer coatings.

Implanted biomaterials can be regarded in a cornerstone in the domain of bone surgery. Their surfaces are expected to fulfil two particular requirements: preventing the settlement and the development of bacteria, and stimulating bone cells in view to foster osseointegration. Therefore, a modern approach consists in the design of dual functional coatings with both antibacterial and osteogenic features. To this end, we developed ultrathin Layer-by-Layer (LbL) coatings composed of biocompatible polyelectrolytes, namely chondroitin sulfate A (CSA) and poly-l-lysine (PLL). The coatings were crosslinked with genipin (GnP), a natural and biocompatible crosslinking agent, to increase their resistance against environmental changes, and to confer them adequate mechanical properties with regards to bone cell behaviors. Antibacterial activity was obtained with nisin Z, an antimicrobial peptide (AMP), which is active against gram-positive bacteria. The coatings had a significant bactericidal impact upon Staphylococcus aureus, with fully maintained bone cell adhesion, proliferation and osteogenic differentiation.