preparation-extracellular-matrix-protein-fibers-for-brillouin

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

University of Exeter

Raman microspectroscopy has been used to investigate the structure of alpha-elastin and fibrous elastin from ligament and aorta, and to explore changes associated with mechanical strain and temperature. Although no vibrational modes associated with cross-linking of the fibers could be identified, the secondary structure of dehydrated fibrous elastin was significantly different from alpha-elastin. The former differed from previous experimental measurements, but was close to the theoretical predictions with 36% beta-structures, 46% unordered, and 18% alpha-helix. Alpha-elastin contained 29% beta-structures, 53% unordered, and 18% alpha-helix. In nuchal fibers the amide I mode was polarized, consistent with the peptide bond. Strains of up to 60% in ligament fiber bundles resulted in no significant shifts in peak position or in secondary structure. Polarization measurements revealed that the peptide bonds and several side chains re-orientated closer to the fiber axis. Heating nuchal fibers to 60 degrees C to increase the energetic component of the elasticity was associated with a 30% increase in the proportion of beta-structures in the amide I band, a 50% increase in the amide III band, and a 50% reduction in the signal from bound water.

The molecular structure and physical properties of elastin fibers as revealed by Raman microspectroscopy.

Raman spectra have been determined for hyaluronan, chondroitin-4-sulfate, chondroitin-6-sulfate, aggrecan monomers and aggregates. The nature of the saccharides and the pattern of sulfation can be discerned. There were only small spectral changes with pH and ionic composition. Differences between hydroxyl vibrations, bulk water and solution conditions are shown. The spectrum of aggrecan is dominated by chondroitin sulfate contribution. The sulfation pattern and ratio of protein to glycosaminoglycan and the secondary structure of the core protein were determined.

Structural analysis of glycosaminoglycans and proteoglycans by means of Raman microspectrometry.

Diabetic retinopathy is one of the leading causes of blindness in the United States and other parts of the world. Historically, laser photocoagulation and vitrectomy surgery have been used for the treatment of diabetic retinopathy, including diabetic macular edema. Both procedures have proven to be useful under certain conditions but have their limitations. New pathways and processes that promote diabetic retinopathy have been identified, and several new therapeutic approaches are under investigation. These new therapies may be beneficial in the treatment of diabetic retinopathy and include antivascular endothelial growth factor agents, corticosteroids, and therapies that may potentially target a number of additional diabetic retinopathy-related factors and processes, including antisense oligonucleotides. Second-generation antisense oligonucleotides, such as iCo-007, may offer a significant advantage in the treatment of diabetic retinopathy by downregulating the signal pathways of multiple growth factors that seem to play a critical role in the process of ocular angiogenesis and vascular leakage. Benefits of such molecules are expected to include the specificity of the kinase target and an extended half-life, resulting in less frequent intravitreal drug administration, resistance to molecule degradation, and a good safety profile.

Antisense oligonucleotide therapy in diabetic retinopathy.

The Patient-Centered Medical Home (PCMH) is a primary care model that provides coordinated and comprehensive care to patients to improve health outcomes. This paper addresses practical issues that arise when transitioning a traditional primary care practice into a PCMH recognized by the National Committee for Quality Assurance (NCQA). Individual organizations' experiences with this transition were gathered at a PCMH workshop in Alexandria, Virginia in June 2010. An analysis of their experiences has been used along with a literature review to reveal common challenges that must be addressed in ways that are responsive to the practice and patients' needs. These are: NCQA guidance, promoting provider buy-in, leveraging electronic medical records, changing office culture, and realigning workspace in the practice to accommodate services needed to carry out the intent of PCMH. The NCQA provides a set of standards for implementing the PCMH model, but these standards lack many specifics that will be relied on in location situations. While many researchers and providers have made critiques, we see this vagueness as allowing for greater flexibility in how a practice implements PCMH.

Lessons learned from implementing the patient-centered medical home.

Stimulated Raman scattering (SRS) has been applied to unstained samples of articular cartilage enabling the investigation of living cells within fresh tissue. Hyperspectral SRS measurements over the CH vibrational region showed variations in protein and lipid content within the cells, pericellular matrix and interterritorial matrix. Changes in the cells and pericellular matrix were investigated as a function of depth into the cartilage. Lipid was detected in the pericellular matrix of superficial zone chondrocytes. The spectral profile of lipid droplets within the chondrocytes indicated that they contained predominantly unsaturated lipids. The mineral content has been imaged by using the PO₄³⁻ vibration at 959 cm⁻¹ and the CO₃²⁻ vibration at 1070 cm⁻¹. Both changes in cells and mineralization are known to be important factors in the progression of osteoarthritis. SRS enables these to be visualized in fresh unstained tissue and consequently should benefit osteoarthiritis research.

Chemically specific imaging and in-situ chemical analysis of articular cartilage with stimulated Raman scattering.

Adipose tissue (AT) expansion in obesity is characterized by cellular growth and continuous extracellular matrix (ECM) remodeling with increased fibrillar collagen deposition. It is hypothesized that the matrix can inhibit cellular expansion and lipid storage. Therefore, it is important to fully characterize the ECM's biomechanical properties and its interactions with cells. In this study, we characterize and compare the mechanical properties of human subcutaneous and omental tissues, which have different physiological functions. AT was obtained from 44 subjects undergoing surgery. Force/extension and stress/relaxation data were obtained. The effects of osmotic challenge were measured to investigate the cellular contribution to tissue mechanics. Tissue structure and its response to tensile strain were determined using nonlinear microscopy. AT showed nonlinear stress/strain characteristics of up to a 30% strain. Comparing paired subcutaneous and omental samples (n = 19), the moduli were lower in subcutaneous: initial 1.6 ± 0.8 (means ± SD) and 2.9 ± 1.5 kPa (P = 0.001), final 11.7 ± 6.4 and 32 ± 15.6 kPa (P < 0.001), respectively. The energy dissipation density was lower in subcutaneous AT (n = 13): 0.1 ± 0.1 and 0.3 ± 0.2 kPa, respectively (P = 0.006). Stress/relaxation followed a two-exponential time course. When the incubation medium was exchanged for deionized water in specimens held at 30% strain, force decreased by 31%, and the final modulus increased significantly. Nonlinear microscopy revealed collagen and elastin networks in close proximity to adipocytes and a larger-scale network of larger fiber bundles. There was considerable microscale heterogeneity in the response to strain in both cells and matrix fibers. These results suggest that subcutaneous AT has greater capacity for expansion and recovery from mechanical deformation than omental AT.

The mechanical properties of human adipose tissues and their relationships to the structure and composition of the extracellular matrix.

Elastin is a major component of tissues such as lung and blood vessels, and endows them with the long-range elasticity necessary for their physiological functions. Recent research has revealed the complexity of these elastin structures and drawn attention to the existence of extensive networks of fine elastin fibres in tissues such as articular cartilage and the intervertebral disc. Nonlinear microscopy, allowing the visualization of these structures in living tissues, is informing analysis of their mechanical properties. Elastic fibres are complex in composition and structure containing, in addition to elastin, an array of microfibrillar proteins, principally fibrillin. Raman microspectrometry and X-ray scattering have provided new insights into the mechanisms of elasticity of the individual component proteins at the molecular and fibrillar levels, but more remains to be done in understanding their mechanical interactions in composite matrices. Elastic tissue is one of the most stable components of the extracellular matrix, but impaired mechanical function is associated with ageing and diseases such as atherosclerosis and diabetes. Efforts to understand these associations through studying the effects of processes such as calcium and lipid binding and glycation on the mechanical properties of elastin preparations in vitro have produced a confusing picture, and further efforts are required to determine the molecular basis of such effects.

The structure and micromechanics of elastic tissue.

Brillouin light scattering (BLS) spectroscopy is a technique that is able to detect thermally excited phonons within a material. The speed of propagation of these phonons can be determined from the magnitude of the Brillouin frequency shift between incident and scattered light, thereby providing a measure of the mechanical properties of the material in the gigahertz range. The mechanical properties of the extracellular matrices of biological tissues and their constituent biopolymers are important for normal tissue function and disturbances in these properties are widely implicated in disease. BLS offers the prospect of measuring mechanical properties on a microscopic scale in living tissues, thereby providing insights into structure-function relationships under normal and pathological conditions. In this study, we investigated BLS in collagen and elastin-the fibrous proteins of the extracellular matrix (ECM). Measurements were made on type I collagen in rat tail tendon, type II collagen in articular cartilage and nuchal ligament elastin. The dependence of the BLS spectrum on fibre orientation was investigated in a backscattering geometry using a reflective substrate. Two peaks, a bulk mode arising from phonon propagation along a quasi-radial direction to the fibre axis and a mode parallel to the surface, depending on sample orientation relative to the fibre axis, could be distinguished. The latter peak was fitted to a model of wave propagation through a hexagonally symmetric elastic solid, and the five components of the elasticity tensor were combined to give axial and transverse Young's, shear and bulk moduli of the fibres. These were 10.2, 8.3, 3.2 and 10.9 GPa, and 6.1, 5.3, 1.9 and 8 GPa for dehydrated type I collagen and elastin, respectively. The former values are close to those previously reported. A microfocused BLS approach was also applied providing selection of single fibres. The moduli of collagen and elastin are much higher than those measured at lower frequency using macroscopic strains, and the difference between them is much less. We therefore believe, like previous investigators, that molecular-scale viscoelastic effects are responsible for the frequency dependence of the fibre biomechanics. Combining BLS with larger-scale mechanical testing methods therefore should, in the future, provide a means of following the evolution of mechanical properties in the formation of the complex structures found in the ECM.

Biomechanics of fibrous proteins of the extracellular matrix studied by Brillouin scattering.

The cyanogen bromide-resistant proteins of lamprey cartilage are biochemically related to the mammalian elastic protein, elastin. This study investigates their mechanical properties and enquires whether, like elastin, long-range elasticity arises in them from a combination of entropic and hydrophobic mechanisms. Branchial and pericardial proteins resembled elastin mechanically, with elastic moduli of 0.13-0.35 MPa, breaking strains of 50%, and low hysteresis. Annular and piston proteins had higher elastic moduli (0.27-0.75 MPa) and larger hysteresis. Exchanging solvent water for trifluoroethanol increased the elastic moduli, whereas increasing temperature lowered the elastic moduli. Raman microspectrometry showed small differences in side-chain modes consistent with reported biochemical differences. Decomposition of the amide I band indicated that the secondary structures were like those of elastin, preponderantly unordered, which probably confer the conformational flexibility necessary for entropy elasticity. Piston and annular proteins showed the strongest interactions with water, suggesting, together with the mechanical testing data, a greater role of hydrophobic interactions in their mechanics. Two-photon imaging of intrinsic fluorescence and dye injection experiments showed that annular and piston proteins formed closed-cell honeycomb structures, whereas the branchial and pericardial proteins formed open-cell structures, which may account for the differences in mechanical properties.

Ellen Green

School of Physics and Astronomy

University of Exeter

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy