Alaa Adawy

Versatile wedge-based system for the construction of unidirectional collagen scaffolds by directional freezing: practical and theoretical considerations

Aligned unidirectional collagen scaffolds may aid regeneration of those tissues where alignment of cells and extracellular matrix is essential, as for instance in cartilage, nerve bundles, and skeletal muscle. Pores can be introduced by ice crystal formation followed by freeze-drying, the pore architecture reflecting the ice crystal morphology. In this study we developed a wedge-based system allowing the production of a wide range of collagen scaffolds with unidirectional pores by directional freezing. Insoluble type I collagen suspensions were frozen using a custom-made wedge system, facilitating the formation of a horizontal as well as a vertical temperature gradient and providing a controlled solidification area for ice dendrites. The system permitted the growth of aligned unidirectional ice crystals over a large distance (>2.5 cm), an insulator prolonging the freezing process and facilitating the construction of crack-free scaffolds. Unidirectional collagen scaffolds with tunable pore sizes and pore morphologies were constructed by varying freezing rates and suspension media. The versatility of the system was indicated by the construction of unidirectional scaffolds from albumin, poly(vinyl alcohol) (a synthetic polymer), and collagen-polymer blends producing hybrid scaffolds. Macroscopic observations, temperature measurements, and scanning electron microscopy indicated that directed horizontal ice dendrite formation, vertical ice crystal nucleation, and evolutionary selection were the basis of the aligned unidirectional ice crystal growth and, hence, the aligned unidirectional pore structure. In conclusion, a simple, highly adjustable freezing system has been developed allowing the construction of large (hybrid) bioscaffolds with tunable unidirectional pore architecture.

A practical kit for micro-scale application of the ceiling crystallisation method

We designed a kit to facilitate the optimisation of protein crystal growth by means of the ceiling crystallisation method. This ceiling kit allows for diffusion-limited crystal growth with a total volume down to 10 μl per trial and air-tight sealing for any type of protein sample. The ceiling crystals grow to sufficient size for diffraction experiments. In this highlight, we fully describe the features of this kit and how easily it can be used to yield protein crystals.

Illuminating protein crystal growth using fluorophore-labelled proteins

Incorporation of trace amounts of fluorophore-labelled proteins is used to study several optical properties and the growth history of protein crystals. Qualitative characterisations of the crystals have been performed by means of confocal laser scanning microscopy and polarisation microscopy. Our results first of all show that labelled proteins do not always act as micro-heterogeneous impurities for the crystals of their parent proteins and may behave differently towards different polymorphs of these protein crystals. Furthermore, labelled proteins may also show a distribution profile very different from their unlabelled counterparts when added as a heterogeneous impurity during crystallisation. Nevertheless, a labelled protein that is incorporated in the growing crystals of its unlabelled counterpart can already provide valuable qualitative information on crystal symmetry and growth history, based upon its orientation, distribution and incorporation efficiency into the host crystals.

The development of the depletion zone during ceiling crystallization: phase shifting interferometry and simulation results

The growth of high quality protein crystals is essential for the determination of their structure. This process is governed by many physical factors such as mass transport and solution flow. The quality of the crystals is usually better under diffusion-limited growth conditions, where a depleted zone of the solution encapsulates the crystal. We developed a Mach–Zehnder-based phase shifting interferometer coupled to image processing software to study the concentration gradients which develop around a crystal during its growth or dissolution. The depletion zones and the diffusion boundary layers around growing and dissolving KH2PO4 crystals are monitored and processed by a MATLAB based algorithm. Our main emphasis was to analyze the ceiling crystallization conditions in which the crystal is placed at the very top of the growth cell and therefore the solute transport is largely diffusion-limited. The experimental results are compared with simulations using finite element-based numerical calculations. The combined results clearly demonstrate the positive effect of the ceiling crystallization approach on crystal growth.

Sub‐Micron Polymeric Stomatocytes as Promising Templates for Confined Crystallization and Diffraction Experiments

The possibility of using sub-micrometer polymeric stomatocytes is investigated to effectuate confined crystallization of inorganic compounds. These bowl-shaped polymeric compartments facilitate confined crystallization while their glassy surfaces provide their crystalline cargos with convenient shielding from the electron beams harsh effects during transmission electron microscopy experiments. Stomatocytes host the growth of a single nanocrystal per nanocavity, and the electron diffraction experiments reveal that their glassy membranes do not interfere with the diffraction patterns obtained from their crystalline cargos. Therefore, it is expected that the encapsulation and crystallization within these compartments can be considered as a promising template (nanovials) that hold and protect nanocrystals and protein clusters from the direct radiation damage before data acquisition, while they are examined by modern crystallography methodologies such as serial femtosecond crystallography.