The Influence of Arginine on the Response of Enamel Matrix Derivative (EMD) Proteins to Thermal Stress: Towards Improving the Stability of EMD-Based Products

In a current procedure for periodontal tissue regeneration, enamel matrix derivative (EMD), which is the active component, is mixed with a propylene glycol alginate (PGA) gel carrier and applied directly to the periodontal defect. Exposure of EMD to physiological conditions then causes it to precipitate. However, environmental changes during manufacture and storage may result in modifications to the conformation of the EMD proteins, and eventually premature phase separation of the gel and a loss in therapeutic effectiveness. The present work relates to efforts to improve the stability of EMD-based formulations such as Emdogain (TM) through the incorporation of arginine, a well-known protein stabilizer, but one that to our knowledge has not so far been considered for this purpose. Representative EMD-buffer solutions with and without arginine were analyzed by 3D-dynamic light scattering, UV-Vis spectroscopy, transmission electron microscopy and Fourier transform infrared spectroscopy at different acidic pH and temperatures, T, in order to simulate the effect of pH variations and thermal stress during manufacture and storage. The results provided evidence that arginine may indeed stabilize EMD against irreversible aggregation with respect to variations in pH and T under these conditions. Moreover, stopped-flow transmittance measurements indicated arginine addition not to suppress precipitation of EMD from either the buffers or the PGA gel carrier when the pH was raised to 7, a fundamental requirement for dental applications.

Characterisation of Living Matter Using Advanced Spectroscopy Techniques

Markus Bonda

In the context of an ageing society neurodegenerative disease have become more and more frequent among humans. Opposing the benefits of a longer life, these diseases have thus triggered research on neurons and how they interact with each other. In vitro as well as in vivo experiments are comparably involved. Due to the complex mechanisms occurring at the neuronal level there is a need for developing suitable techniques. Such techniques are required to provide high resolution in several respects in order to track cellular properties: Fourier transform infrared spectroscopy (FTIR) along with high-brilliance synchrotron radiation matches the requirements of high spectral and spatial resolution for chemical distinction perfectly. We have investigated a rat model of Huntington's disease (HD) with synchrotron-assisted Fourier transform infrared microspectroscopy (SIRMS). Several neurodegenerative diseases, ranging from Alzheimer's to prion disease, have now been identified to be caused by proteins which auto-assemble into high molecular weight amyloid aggregates or fibrils. HD is related to abnormal polyglutamine (polyQ) repeats. Its symptoms are progressive deterioration of cognitive and motor functions, along with extensive loss of neurons. In our model, one brain hemisphere was infected with HD while keeping the second one as a control. The two types of matter in the striatum are affected in different manners: in neuron-rich gray matter exposed to the disease, a higher content of aggregated protein is detected, but no signs of cell death. In contrast, myelin-rich white matter did not show any aggregates, but surprisingly showed a significant increase in phosphorylation. We interpret this result as the activation of the cellular response to stress which leads in the end to cell death. The drastic changes in the white matter were detected in the case of multiple sclerosis. We have studied its animal model, the experimental autoimmune encephalomyelitis (EAE). EAE is characterized by heavy loss of the insulating lipid-layer, the myelin around axons. This has been clearly confirmed by SIRMS and FTIR. Analysis of spatially resolved maps by both unsupervised principal component analysis and chemical signatures showed additional features of EAE. Both studies indicate that SIRMS is a powerful tool of detecting complex chemical processes in biological tissue in a relatively easy way. In addition to nanometer spatial resolution, the photonic force microscope (PFM) provides microsecond temporal resolution. A PFM tracks the Brownian motion of micron-sized beads embedded in solutions. Thanks to the high resolutions, we can calculate velocity auto-correlation functions (vacf) of the beads that are extremely sensitive on the short-time scale. We found that they depend in solutions containing polymers on the mesh size. The narrower the polymer mesh size, the earlier perturbations occur in the bead's motion. Polymer solutions possess a very interesting feature which is their viscoelasticity. Based on the mean square displacements of the beads, the complex shear modulus is obtained describing in a frequency-dependent manner viscosity and elasticity. In this way we could distinguish different polymers and also a pathologically relevant fibril: α-synuclein involved in Parkinson's disease.

EPFL2011

Characterisation of Living Matter Using Advanced Spectroscopy Techniques

Markus Bonda

EPFL2011