Chain dynamics of human dermis by Thermostimulated currents: A tool for new markers of aging.

BACKGROUND/PURPOSE: The purpose of this clinical study was to identify dielectric markers to complete a previous thermal and vibrational study on the molecular and organizational changes in human dermis during intrinsic and extrinsic aging. METHODS: Sun-exposed and non-exposed skin biopsies were collected from 28 women devised in two groups (20-30 and ≥60 years old). The dielectric relaxation modes associated with localized and delocalized dynamics in the fresh and dehydrated state were determined by the Thermostimulated currents technique (TSC). RESULTS: Intrinsic and extrinsic aging induced significant evolution of some of the dielectric parameters of localized and delocalized dynamics of human skin. With photo-aging, freezable water forms a segregated phase in dermis and its dynamics is close to free water, what evidences the major role of extrinsic aging on water organization in human skin. Moreover, TSC indicators highlight the restriction of localized mobility with intrinsic aging due to glycation, and the cumulative effect of chronological aging and photo-exposition on the molecular mobility of the main structural proteins of the dermis at the mesoscopic scale. CONCLUSION: TSC is a well-suited technique to scan the molecular mobility of human skin. It can be uses as a relevant complement of vibrational and thermal characterization to follow human skin modifications with intrinsic and extrinsic aging.

Identification of ageing biomarkers in human dermis biopsies by thermal analysis (DSC) combined with Fourier transform infrared spectroscopy (FTIR/ATR).

BACKGROUND/PURPOSE: The purpose of this clinical study was to identify suitable biomarkers for a better understanding of the molecular and organizational changes in human dermis during intrinsic and extrinsic ageing. METHODS: Sun-exposed and non-exposed skin biopsies were collected from twenty-eight women devised in two groups (20-30 and ≥60 years old). The hydric organization and thermal transitions were determined by Differential Scanning Calorimetry (DSC). Fourier Transform Infrared spectroscopy (FTIR) was used to identify the absorption bands of the dermis and to quantify the different absorbance ratio. RESULTS: The amounts of total, freezable and unfreezable water were determined. A significant increasing amount of freezable water is evidenced in sun-exposed area skin of aged group compared with young group (P=.0126). Another significant effect of extrinsic ageing (P=.0489) is the drastic decrease of fibrillary collagen, the main protein component of dermis. The only significant effect of intrinsic ageing (P=.0184) is an increase of the heat-stable fraction of collagens in dermis. CONCLUSION: DSC and FTIR are well-suited techniques to characterize human skin, giving accurate results with a high reproducibility. The combination of these techniques is useful for a better understanding of human skin modifications with intrinsic and extrinsic ageing.

Conformational and thermal characterization of left ventricle remodeling post-myocardial infarction.

Adverse cardiac remodeling after myocardial infarction (MI) causes impaired ventricular function and heart failure. Histopathological characterization is commonly used to detect the location, size and shape of MI sites. However, the information about chemical composition, physical structure and molecular mobility of peri- and infarct zones post-MI is rather limited. The main objective of this work was to explore the spatiotemporal biochemical and biophysical alterations of key cardiac components post-MI. The FTIR spectra of healthy and remote myocardial tissue shows amides A, I, II and III associated with proteins in freeze-died tissue as major absorptions bands. In infarcted myocardium, the spectrum of these main absorptions was deeply altered. FITR evidenced an increase of the amide A band and the distinct feature of the collagen specific absorption band at 1338cm-1 in the infarct area at 21days post-MI. At 21days post-MI, it also appears an important shift of amide I from 1646cm-1 to 1637cm-1 that suggests the predominance of the triple helical conformation in the proteins. The new spectra bands also indicate an increase in proteoglycans, residues of carbohydrates in proteins and polysaccharides in ischemic areas. Thermal analysis indicates a deep increase of unfreezable water/freezable water in peri- and infarcted tissues. In infarcted tissue is evidenced the impairment of myofibrillar proteins thermal profile and the emergence of a new structure. In conclusion, our results indicate a profound evolution of protein secondary structures in association with collagen deposition and reorganization of water involved in the scar maturation of peri- and infarct zones post-MI.

Conformational and thermal characterization of a synthetic peptidic fragment inspired from human tropoelastin: Signature of the amyloid fibers.

OBJECTIVES: This work deals with the conformational and thermal characterization of a synthetic peptide (S4) released during the proteolysis of human tropoelastin by the matrix metalloproteinase-12 that was shown to form amyloid-like fibres under certain conditions. MATERIALS AND METHODS: S4 peptides were synthesized by solid-phase methodology and aggregated in solution at 80°C. Fourier transform-infrared spectroscopy (FT-IR) was used to access the secondary structure. Thermal characterization was performed by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). RESULTS: The DSC study of the soluble linear peptide S4 in solution in TBS reveals the irreversible aggregation into amyloid fibres. FT-IR, DSC and TGA analyses performed on freeze-dried samples evidence differences between the linear peptide and its associated amyloid-like fibres, both on the conformation and the physical structure. When S4 peptides are aggregated, the prominent conformation scanned by FT-IR is the cross ß-structure, corresponding to TGA to an increase of the thermal stability. Moreover, the DSC thermograms of S4 fibres are characteristic of a highly ordered structure, in contrast to the DSC thermograms of S4 linear peptides, characteristic of an amorphous structure. Finally, the DSC analysis of differently hydrated S4 fibres brings to the fore the specific thermal answer of the wet interfaces of the cross ß-fibres. CONCLUSION: FT-IR and thermal techniques are well suited to evidence conformational and structural differences between the soluble peptide and its amyloid form.

Analysis of the molecular mobility of collagen and elastin in safe, atheromatous and aneurysmal aortas.

AIM OF THE STUDY: In this study, we propose to use a thermal technique, Differential Scanning Calorimetry (DSC) to follow the evolution of elastin and collagen in safe and pathological cardiovascular tissues. PATIENTS AND METHODS: The first part of this study deals with the analysis of the elastin network and associated proteins during ageing (from children to old persons) in aortic walls. The second part is devoted to the characterization of the collagenic phase in aneurysms. In both cases, physical data are correlated with biochemical analyses. RESULTS AND CONCLUSION: For old persons aortas with atheromatous stades, elastin and associated proteins are found to interpenetrate to form a homogenous phase. Abdominal aortic aneurysms (AAA) are characterized by structural alterations of the aortic wall resulting from the degradation of elastic fibers and an increase of collagen/elastin ratio. Notable modifications are evidenced between collagen from control tissue and collagen from AAA, particularly concerning the thermal denaturation. Biochemical and thermal results are compatible with the increase of new collagen deposition and/or impairment of the collagen phase stability in the extracellular matrix of AAAs.

Human tropoelastin sequence: dynamics of polypeptide coded by exon 6 in solution.

Calorimetric studies were performed on exon 6 in powdered form and in solution [water and 2,2,2-trifluoroethanol (TFE), a structure-inducing solvent or cosolvent]. Dynamic dielectric spectroscopy (DDS) analyses were realized in water and 20% TFE. The major role of solvent-peptide organization is evidenced with these techniques. Calorimetric measurements reveal the structural water organization around the polypeptide as well as the presence of hydrophobic interactions in TFE solution. Dielectric measurements showed for exon 6/water a decrease of relaxations times of bulk solvent implying a faster dynamics with a slight increase of the activation entropy, suggesting that exon 6 probably creates disorder within the solvent. For TFE/water mixtures, an influence of exon 6 on its environment was seen with a relaxation associated with the exon 6/solvent interactions reinforced by storage of 72 h. Finally, exon 6/solvent interactions were clearly observed with addition of TFE.

Comparison of chemical treatments on the chain dynamics and thermal stability of bovine pericardium collagen.

A new approach for the replacement of heart valves consists of obtaining an acellular matrix from animal aortic valves that performs mechanically, is nonantigenic, and is free from calcification and fibroblast proliferation. Novel biochemical treatments must be developed for this purpose. In this work, we focus on the characterization of collagen in acellular bovine cardiovascular tissues, fresh or glutaraldehyde treated, and stored in different solutions [phosphate-buffered saline (PBS), ethanol, octanol, and glutaraldehyde], to determine whether the resulting fibrous material is structurally preserved. The preservation of the triple helical structure of collagen is checked by differential scanning calorimetry (DSC), which is a well suited technique to analyze thermal transitions in proteins, such as denaturation. To get insight into the molecular dynamics of collagen in the nanometric range, we used thermally stimulated currents, a dielectric technique running at low frequency, that measure the dipolar reorientations in proteins submitted to a static electrical field. The combined use of these two techniques allowed us to evaluate the physical structure and conformation of collagen after the different chemical treatments. We have found that the glutaraldehyde treatment followed by octanol storage preserves the triple helical conformation of the polypeptidic chains of collagen, contrary to the ethanol and PBS storage that induce drastic changes in the thermal and dielectric behavior of the protein. Moreover, this particular chemical treatment stabilizes the collagen structure (shift toward high temperature of the collagen denaturation and stiffening of the chains by a cross-linking action) when compared to the control sample, and so could provide interesting fibrous material for the conception of bioprosthetic heart valve.

Caracterización y biocompatibilidad de las prótesis vasculares de poliuretano estabilizado con polidimetilsiloxano / Characterization and biocompatibility of vascular prostheses made of polyurethane stabilized with polydimethylsiloxane

Introducción. El desarrollo de nuevos biomateriales ha desembocado en la aparición de nuevas prótesis vasculares que mejoren el comportamiento de injertos protésicos de pequeño calibre. Objetivo. El objetivo del presente trabajo es el estudio del comportamiento biológico de prótesis vasculares de poliuretano. Material y métodos. Prótesis: poliuretano-polidimetilsiloxano (PU-PDMS). Caracterización: fragmentos de PU-PDMS se procesaron para su estudio en microscopia óptica y electrónica de barrido. Se determinó la carga eléctrica de la superficie interna mediante análisis espectral. Biocompatibilidad: fragmentos (1 cm2) de PU-PDMS se implantaron en el músculo dorsal de conejos Nueva Zelanda (n= 18) durante 3 y 8 meses. Realizamos estudios morfológicos, inmunohistoquímicos (antiactina) y de reacción de cuerpo extraño (RAM11). Siembra: fragmentos de 1 cm2 se sembraron con células endoteliales de vena umbilical humana. Tiempos de estudio: 24, 48, 72 horas y 7 días. Resultados. La composición es fibrilar, con presencia de numerosos poros. Existencia de cargas negativas en la superficie interna del biomaterial. A los tres meses, la prótesis se embebe en tejido neoformado muy vascularizado y rico en células blancas y células de reacción a cuerpo extraño. A los 8 meses se puede observar la total integración del biomaterial, que aparece rodeado de colágeno y muy vascularizado. A las 24 horas de la siembra observamos una superficie endotelizada, que deja al descubierto grandes poros que se tapizan en los estadios posteriores. Conclusiones. Las prótesis PUPDMS presentan características adecuadas para utilizarse como sustitutos vasculares, gracias a su estructura, ausencia de rechazo y buena integración a corto y medio plazo. (AU)

Alterations in the chain dynamics of insoluble elastin upon proteolysis by serine elastases.

The high temperature dielectric relaxations of purified and elastolized ligamentum nuchae elastin in the dry state have been investigated by thermally stimulated depolarization current spectrometry, with an equivalent frequency comprised between 10(-2) and 10(-3) Hz. A main relaxation mode, located close to 150 degrees C and attributed to the dielectric manifestation of a glass transition, is found for all samples. After decomposition by the fractional polarization method, the analysis of the high temperature mode shows the existence of two relaxation mechanisms: a cooperative one, associated with flexible zones of the protein, and an isoenthalpic one, corresponding to more ordered and constrained zones. The activation parameters of the two mechanisms are dependent on the extent of elastolysis and on the nature of enzyme (pancreatic elastase vs leukocyte elastase). Both enzymes influence the dielectric behavior of elastin in a similar way: the activation enthalpy maximum of the relaxing units located in the flexible zones, characteristic of the cooperative length, decreases with increasing hydrolysis. Moreover, the isoenthalpic mechanism becomes cooperative at the highest extent of elastolysis, which highlights release of constraints in ordered zones. Nevertheless, the differences found between the two enzymatic hydrolyses are characteristic of distinct sites of cleavage in the elastin network.

Dielectric characterization of collagen, elastin, and aortic valves in the low temperature range.

The low temperature dielectric relaxation of porcine aortic valves and its main macromolecular proteins. i.e. elastin and collagen, have been investigated in the dry state and at low levels of hydration by thermally stimulated currents spectrometry, with an equivalent frequency of 10(-3) Hz. Two secondary relaxation modes, labeled gamma and beta with increasing temperature, are found for the three materials. Since the gamma-mode is independent upon hydration while the beta-mode is strongly plasticized by water, these relaxation modes have been attributed to localized motions of the polypeptidic chains containing apolar and polar residues, respectively. The deconvolution of the beta-mode by fractional polarization gives the experimental distribution of the dielectric relaxation times of the three materials, and allows us to deduce the activation parameters of each elementary process. These analyses shows the existence of compensation phenomena between the activation parameters, implying cooperative mechanisms. The occurrence of these phenomena with their characteristic parameters are used to specify the origin of the localized relaxation modes in collagen and elastin, and to assign the specific role of each protein in the aortic valves.

Characterisation of elastin and collagen in aortic bioprostheses.

Porcine aortic valves used as cardiac valve bioprostheses are well adapted to physiological functions in the short term, but they lack long-term durability. Several multi-step extractions have been performed to obtain a perfectly acellular matrix. A new physical methodology is proposed to evaluate the resulting fibrous protein damage after biochemical extraction (TRI-COL and SDS). Thermal analysis techniques are adapted to collagen and elastin characterisation in the solid state. The aortic tissue thermal transitions are determined by differential scanning calorimetry (DSC): elastin glass transition is observed around 200 degrees C, and collagen denaturation is observed around 230 degrees C. These parameters are characteristic of the elastin network arrangement and of collagen triple-helix stability. The technique of thermostimulated currents (TSC) is well suited to specify the chain dynamics of proteins. The low-temperature relaxations observed in both collagen and elastin are associated with localised motions, whereas the high-temperature modes are attributed to more delocalized motions of the chains. Therefore TSC and DSC spectrometries allow physical parameters specific to collagen and elastin to be obtained and their interaction in aortic tissues to be determined. According to the significant evolution of these parameters on SDS samples, the destabilizing effect of this detergent is highlighted.

Thermal analysis characterization of aortic tissues for cardiac valve bioprostheses.

Two multistep extractions were achieved on porcine aortic tissues to obtain acellular matrices used for cardiac bioprostheses. The evaluation of structural modifications and the possible damage of extracellular matrix fibrous proteins were investigated by means of thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Protein-water interactions and degradation temperatures were determined by TGA. DSC was used to characterize protein thermal transitions (glass transition and denaturation), which provided information on the dynamic structure of the aortic tissue components. Sodium dodecyl sulfate (SDS) extraction had a destructuring effect, while Triton and cholate treatments did not affect the structural integrity of either elastin and collagen. A DSC comparison showed that SDS destabilizes the collagen triple helical domain and swells the elastin network.