Mechanical Properties and Functions of Elastin: An Overview.

Human tissues must be elastic, much like other materials that work under continuous loads without losing functionality. The elasticity of tissues is provided by elastin, a unique protein of the extracellular matrix (ECM) of mammals. Its function is to endow soft tissues with low stiffness, high and fully reversible extensibility, and efficient elastic-energy storage. Depending on the mechanical functions, the amount and distribution of elastin-rich elastic fibers vary between and within tissues and organs. The article presents a concise overview of the mechanical properties of elastin and its role in the elasticity of soft tissues. Both the occurrence of elastin and the relationship between its spatial arrangement and mechanical functions in a given tissue or organ are overviewed. As elastin in tissues occurs only in the form of elastic fibers, the current state of knowledge about their mechanical characteristics, as well as certain aspects of degradation of these fibers and their mechanical performance, is presented. The overview also outlines the latest understanding of the molecular basis of unique physical characteristics of elastin and, in particular, the origin of the driving force of elastic recoil after stretching.

DSC, FT-IR and NIR with Chemometric Assessment Using PCA and HCA for Estimation of the Chemical Stability of Oral Antidiabetic Drug Linagliptin in the Presence of Pharmaceutical Excipients.

Pharmaceutical excipients should not interact with active substances, however, in practice, they sometimes do it, affecting the efficacy, stability and safety of drugs. Thus, interactions between active substances and excipients are not desirable. For this reason, two component mixtures of oral antidiabetic drug linagliptin (LINA) with four excipients of different reactivity, i.e., lactose (LAC), mannitol (MAN), magnesium stearate (MGS) and polyvinylpyrrolidone (PVP), were prepared in a solid state. A high temperature and a high humidity of 60 °C and 70% RH, respectively, were applied as stressors in order to accelerate the potential interactions between LINA and excipients. Differential scanning calorimetry (DSC) as well as Fourier transform infrared (FT-IR) and near infrared (NIR) spectroscopy were used to estimate the changes due to potential interactions. In addition, chemometric computation of the data with principal component analysis (PCA) and hierarchical cluster analysis (HCA) was applied to adequately interpret the findings. Of the excipients used in the present experiment, all of them were not inert in relation to LINA. Some of the interactions were shown without any stressing, whereas others were observed under high-temperature/high-humidity conditions. Thus, it could be concluded that selection of appropriate excipients for LINA is very important question to minimize its degradation, especially when new types of formulations with LINA are being developed and manufactured.

Teicoplanin-Modified HPLC Column as a Source of Experimental Parameters for Prediction of the Anticonvulsant Activity of 1,2,4-Triazole-3-Thiones by the Regression Models.

The cell membrane is a complex system that consists of lipids, proteins, polysaccharides, and amphiphilic phospholipids. It plays an important role in ADME processes that are responsible for the final pharmaceutical effects of xenobiotics (bioavailability, activity). To study drug-membrane interaction at the molecular level, several high-performance liquid chromatography (HPLC) membrane model systems have been proposed which are mimicking mainly its lipid character. The aim of this work was to study interactions of new synthesized antiepileptic compounds of 4-alkyl-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione derivatives with Chirobiotic column containing glycoprotein ligand attached to the silica matrix. The affinity of the analytes to immobilized glycoprotein ligand was examined chromatographically in reversed-phase mode. The thermodynamics of interactions between bioactive compounds and teicoplanin was studied in terms of the van't Hoff linear relationship ln k vs. 1/T in the range of 5-45 °C. Change in enthalpy (ΔH°), change in entropy (ΔS°) and change in Gibbs free energy (ΔG°) were estimated utilizing graphical extrapolation and interpolation methods. The density functional theory (DFT) approach and docking simulations were used to get the molecular interpretation and prove the obtained experimental results. Cross-correlations of chromatographic and thermodynamic parameters with non-empirical topological and quantum chemical indices suggest that the polarizability of analytes appears to be responsible for the interactions of the tested molecules with teicoplanin and, ultimately, their retention on the column. Experimental and theoretical parameters were subjected to statistical analysis using regression models. Partial least squares (PLS) regression model showed the usefulness of the experimentally measured parameter φ0 (MeOH) to discriminate between anticonvulsant active and inactive 1,2,4-triazole-3-thione derivatives. Obtained results point out the usefulness of interaction of potential anticonvulsants with glycoprotein class of compounds to anticipate their activity.

Thermodynamic study of new antiepileptic compounds by combining chromatography on the phosphatidylcholine biomimetic stationary phase and differential scanning calorimetry.

Liquid chromatography coupled to spectrophotometric detection of new antiepileptic compounds, 1,2,4-triazole-3-thione derivatives, on immobilized artificial membrane phosphatidylcholine is reported. The curves representing the relationship between ln k versus 1/T generated under isocratic conditions by the use of methanol and acetonitrile-containing eluent systems have been constructed in order to determine the thermodynamic parameters: the enthalpies, entropies and the relative free energies. The hydrocarbon chains of analytes significantly influenced the membrane behavior of the whole molecules. Excellent correlations of the theoretical lipophilicity with the experimental thermodynamic descriptors, have confirmed contribution of the hydrophobic interactions in the retention process. However, presence of sulfur or oxygen as heteroatoms at R1 substituents in the 1,2,4-triazole ring appears to be responsible for more pronounced selectivity of these compounds on the phosphatidylcholine stationary phase. Molecular dynamics simulations revealed the selective preferences of the phosphatidylcholine with respect to the compounds with either ether of sulfide moieties. Experimental and theoretical set-ups resulted in corresponding outcomes.

High-performance liquid chromatography thermodynamic study of new potential antiepileptic compounds on a cholesterol column using isocratic elution with methanol/water and acetonitrile/water eluent systems.

Basic thermodynamic functions responsible for retention of new 1,2,4-triazole derivatives exhibiting varied antiepileptic activity on cholesterol-based stationary phase were determined. Evaluation of the Gibbs energy change, the change in enthalpy and the change in entropy was based on the van't Hoff relationship representing lnk versus 1/T. A detailed discussion of the van't Hoff equation, exploring the influence of the phase ratio on deviations from linearity in a van't Hoff plot is presented. We show chromatographic evidence to the question of how a varied mobile phase composition may cause different thermodynamic phase ratios. The analysis of data from a differential scanning calorimetry excluded any phase transitions of either the individual solutes or cholesterol stationary phase suspended in the mobile phase components within the studied temperature range.

Effect of glucose on fatigue-induced changes in the microstructure and mechanical properties of demineralized bovine cortical bone.

PURPOSE: The aim of this study was to test a hypothesis that fatigue-induced weakening of cortical bone was intensified in bone incubated in glucose and that this weakening is revealed in the microstructure and mechanical competence of the bone matrix. METHODS: Cubic specimens of bovine femoral shaft were incubated in glucose solution (G) or in buffer (NG). One half of G samples and one half of NG were axially loaded in 300 cycles (30 mm/min) at constant deformation (F); the other half was a control (C). Samples from each group (GF, NGF, GC, NGC) were completely demineralized. Slices from demineralized samples were used for microscopic image analysis. A combined effect of glycation and fatigue on demineralized bone was tested in compression (10 mm/min). Damage of samples during the test was examined in terms of acoustic emission analysis (AE). RESULTS: During the fatigue procedure, resistance to loading in glycated samples decreased by 14.5% but only by 8.1% in nonglycated samples. In glycated samples fatigue resulted in increased porosity with pores significantly larger than in the other groups. Under compression, strain at failure in demineralized bone was significantly affected by glucose and fatigue. AE from demineralized bone matrix was considerably related to the largest pores in the tissue. CONCLUSIONS: The results confirm the hypothesis that the effect of fatigue on cortical bone tissue was intensified after incubation in glucose, both in the terms of the mechanical competence of bone tissue and the structural changes in the collagenous matrix of bone.

The effect of excipients on the release kinetics of diclofenac sodium and papaverine hydrochloride from composed tablets.

For increased analgesic effect, new composed tablets containing diclofenac sodium (DIC) with an addition of papaverine hydrochloride (PAP) were prepared to investigate the mechanism of release of the active substances from tablets with different excipients in eight different formulations. To detect the possible interactions between active substances and excipients differential scanning calorimetry (DSC) was used. A shift of the melting point and enthalpy values of the physical mixtures of tablets components suggested a kind of interaction between components in certain formulations, however, the tabletting process was not disturbed in any of them. Kinetics of drug release from formulations was estimated by zero order, first order and Higuchi and Korsmeyer-Peppas models using results of dissolution of DIC and PAP from tablets. The study revealed that the mechanism of release of active substances was dependent on the excipients contained in tablets and the best fitted kinetics models were obtained for formulations with potentially prolonged release of DIC and PAP.

Does thermodynamic stability of peritoneal collagen change during laparoscopic cholecystectomies? A differential scanning calorimetry (DSC) study.

BACKGROUND: Carbon dioxide pneumoperitoneum used during laparoscopic surgeries alters the integrity of the peritoneum and results in denudation of the basal lamina that might cause altered immune response, inhibited fibrinolysis, hypoxia, and acidosis. The changes in the structure of pneumoperitoneum were described as bulging of mesothelial cells, irregular cell junction's cell membrane degradation, and mesodermal edema. As denaturation of peritoneal proteins reflects overall condition of its structure and interactions with the surrounding molecules, the physical status of collagen was assessed on the basis of parameters of thermal denaturation measured by DSC method. METHODS: Twenty-four female patients operated on due to cholelithiasis were enrolled in this study. Laparoscopic cholecystectomy was performed using standard four-trocar technique, and standard values of insufflated carbon dioxide pneumoperitoneum were used. After trocar placement, the first collection of peritoneal sample (sample A) was performed. The second peritoneal sample (sample B) was collected after the removal of gall bladder. Differential scanning calorimetry (Q200 calorimeter, TA Instruments) was performed on samples defrosted at room temperature. RESULTS: In all samples of peritoneum, a nonreversible endothermal process recognized as denaturation was observed. Sample B obtained at the end of surgery did not differ from sample A obtained at the beginning in terms of all parameters under study. Temperature of denaturation in A and B was correlated only marginally, but enthalpy and specific heat were significantly correlated. The analysis of data from DSC measurements did not reveal differences in physical stability of collagen in peritoneal samples obtained at the beginning and at the end of surgery. Significant negative correlations between duration of CO2 pneumoperitoneum and enthalpy of denaturation in sample B were found. CONCLUSIONS: Differences in enthalpy of denaturation may reflect a quantitative relation between amount of native collagen molecules in the sample and other, non-collagenous components or impaired collagen.

Effects of fatigue on microstructure and mechanical properties of bone organic matrix under compression.

The aim of the study was to investigate whether a fatigue induced weakening of cortical bone was revealed in microstructure and mechanical competence of demineralized bone matrix. Two types of cortical bone samples (plexiform and Haversian) were use. Bone slabs from the midshaft of bovine femora were subjected to cyclical bending. Fatigued and adjacent control samples were cut into cubes and demineralized in ethylenediaminetetraacetic acid. Demineralized samples were either subjected to microscopic quantitative image analysis, or compressed to failure (in longitudinal or transverse direction) with a simultaneous analysis of acoustic emission (AE). In fatigued samples porosity of organic matrix and average area of pores have risen, along with a change in the pores shape. The effect of fatigue depended on the type of the bone, being more pronounced in the plexiform than in Haversian tissue. Demineralized bone matrix was anisotropic under compressive loads in both types of cortical structure. The main result of fatigue pretreatment on mechanical parameters was a significant decrease of ultimate strain in the transverse direction in plexiform samples. The decrease of strain in this group was accompanied by a considerable increase of the fraction of large pores and a significant change in AE energy.

Chemometric processing of pharmaceutical essential oil fingerprints -- comparison of GC, HPLC, TLC, IR spectroscopy, and differential scanning calorimetry.

Fifteen essential oils of pharmaceutical grade were fingerprinted by five techniques: TLC, GC, HPLC, attenuated total reflectance FTIR spectroscopy, and differential scanning calorimetry (DSC). Denoising and baseline removal was found to be a crucial step for correct comparative analysis. Standardization of the signal was not necessary in the presented case; however, it should be considered and checked in each case. Due to small variance explained by first two principal components (below 50%) and outlying observations, the main analysis was performed by Euclidean dendrograms. It was found that almost all techniques besides DSC find real chemical similarities; however, DSC can be used as an additional tool. The similarities among the five techniques were also compared and discussed.

Effect of "in vitro" induced glycation on thermostability of bone tissue.

The aim of the study was to test the hypothesis that glycation would influence thermal stability of bone tissue collagen. Bone samples were incubated in buffer or in ribose solution. Then, half of the ribosylated and half of the control samples were completely demineralized in formic acid. Differential scanning calorimetry was performed for temperatures from 40 °C to 220 °C in nitrogen atmosphere on intact (mineralized) and demineralized bone samples, partially dehydrated at room temperature. Samples were thermally active in temperatures from 110 °C to 210 °C. Few endotherms of a complex nature were found in demineralized and intact bone. Thermodynamics of collagen conformations was affected by glycation, especially in demineralized bone where a significant increase of denaturation temperature (by 3-4 °C) and enthalpy drop (above 20%) were stated after glycation.

Effects of nonenzymatic glycation on mechanical properties of demineralized bone matrix under compression.

PURPOSE: Effects of in vitro induced nonenzymatic glycation of bone collagen on stiffness and fracture of demineralized bone matrix in unconfined compression were investigated. MATERIAL AND METHODS: Regular specimens from mid-shaft of bovine femur were grouped in pairs. One sample (R) from each pair was incubated in ribose solution, control samples (C) were incubated in a buffer solution. Samples were then demineralized in formic acid. Demineralized samples were axially compressed to failure (0.033/s). Direction of compression was along the longitudinal axis of femur (L) or perpendicular (transverse) to that (T). Mechanical behavior of demineralized samples was characterized in terms of secant modulus, stress, and strain at fracture and work to failure. The development of damage was examined in terms of acoustic emission (AE) signal recorded during loading. RESULTS: In L direction, strain at fracture following glycation was lower than in the controls (p=0.038); secant modulus and ultimate stress were not significantly different in R and C. In the transverse direction, strain at fracture in R was higher than in C (p=0.053), as well as work to failure (p=0.020). Anisotropy of bone matrix, defined in terms of a ratio of the parameters in two perpendicular directions, decreased markedly in ribosylated samples. Both the number of AE events and cumulative AE energy during deformation were significantly higher in ribosylated samples than in the control for both directions of compression. CONCLUSION: The study demonstrated that nonenzymatic glycation plays a significant role in modifying organic matrix properties in cortical bone.

Anisotropy of demineralized bone matrix under compressive load.

Two groups of cubic specimens from diaphysis of bovine femur, intact and completely demineralized, were axially compressed. One half of the samples from each group were loaded along the axis of the femur (L) and the other - perpendicularly (T). Intact samples were characterized in terms of elastic modulus; for demineralized samples secant modulus of elasticity was calculated. During compression an acoustic emission (AE) signal was recorded and AE events and energy were analyzed. Samples of intact bone did not reveal any anisotropy under compression at the stress of 80 MPa. However, AE signal indicated an initiation of failure in samples loaded in T direction. Demineralized samples were anisotropic under compression. Both secant modulus of elasticity and AE parameters were significantly higher in T direction than in L direction, which is attributed to shifting and separation of lamellae of collagen fibrils and lamellae in bone matrix.

Evaluation of peritoneal tissue by means of differential scanning calorimetry (DSC).

Abdominal surgeries alter the integrity of the peritoneal layer and cause imbalances among immunological, inflammatory and angiogenic mechanisms within the tissue. During laparoscopic procedures a protective function of the peritoneal layer can be disturbed by the gas used to create a pneumoperitoneum. The aim of this study was to characterize peritoneal tissue by means of differential scanning calorimetry (DSC) as a reference for future investigations on the influence of surgical procedures on the physicochemical state of the peritoneum. Thirty-seven patients participated in the study. Patients were divided into three groups according to the type of surgery: group H - patients who underwent hernia repair; group Ch - patients who underwent laparoscopic cholecystectomy; and group C - patients operated due to rectal cancer. It was observed that onset temperature (T(o)), denaturation temperature (T(m)) and change of enthalpy (ΔH) during thermal denaturation of peritoneal collagen in were significantly different for these three groups of patients. The mean values of onset temperature (T(o)) and denaturation temperature (T(m)) in group H were significantly lower, while DH in this group was significantly higher than in the two other groups (Ch and C). This preliminary study does not answer whether the differences in collagen denaturation found in peritoneal tissue from different groups of patients resulted from a different inherent state of the tissue, or from surgical procedures. However, the results suggest that DSC is an appropriate method to study subtle changes in the physicochemical condition of the peritoneum using small samples obtained during surgical procedures.

Thermostability of bone tissue after immobilization induced osteopenia in a rat model.

Immobilization of load-bearing bones results in imbalance of bone turnover followed by bone loss and impairment of its mechanical function. The question is whether immobilization induced bone loss is accompanied by deterioration of properties of the bone tissue components. Thermally induced transformations of collagen reflect the overall condition of the structure and cross-links in collagen network. The aim of the present study was to investigate whether immobilization induced osteopenia effects stability of collagen in bone tissue. Bone loss was developed by unilateral hindlimb immobilization in adult rats. Effects of unloading on cortical tissue from tibiae were studied after three weeks of unloading (I3R0) and four weeks after remobilization and free convalescence (I3R4) in both tibiae. Thermodynamic parameters of collagen degradation in bone were determined from differential scanning calorimetry (DSC) analysis of partially dehydrated cortical bone samples from tibiae in the range of temperatures from 60 degrees C up to 300 degrees C. All bone samples were thermally very stable showing first clear endothermal process with a peak temperature within a range from 150 degrees C to 169 degrees C, for different samples. The next endotherm, wider and flatter, was observed between 245-298 degrees C with a peak at 255 degrees C - 260 degrees C. There were significant side-to-side (right to left) differences for both endothermal processes in tibiae samples from experimental groups: I3R0 and I3R4. Immobilization of load-bearing bones influences stability of collagen in bone tissue. Free remobilization was not sufficient for recovery of thermal parameters of bone.

Three-point bending and acoustic emission study of adult rat femora after immobilization and free remobilization.

The experiment concerned effects of immobilization and remobilization on mechanical properties of femoral shaft. Twenty-four weeks old male rats were used: two groups (I3 and I3R4) with the right hindlimb immobilized for 3 weeks by taping, and one control (C). In I3R4 immobilization was followed by 4 weeks of free remobilization. Mechanical properties in three-point bending, mass, geometry, and mineralization of bone tissue were measured post mortem in both femora in I3 and I3R4 and in right femora in control. Acoustic emission signals (AE) were recorded during the bending test. The right femora in I3, I3R4 and C did not differ significantly in size, mass and mineralization (ANOVA). The differences were significant considering mechanical parameters and AE signals. In I3 yield bending moment and stiffness were lower (p=0.013 and 0.025) and deflection was larger (p=0.030) than in C. In I3R4 maximum bending moment, yield moment, stiffness and work to failure were lower than in C (p=0.013, 0.009, 0.032, and 0.005). Paired t-test showed that remobilization resulted in worsening of properties of right femora. Side-to-side differences in I3R4 were more pronounced than in I3. Moreover, AE signals from the right femora were more numerous and burst type than from the left. The results demonstrate that strength of bone decreases during the first period of free remobilization. The decrease is accompanied by a significant decrease of bone toughness. The AE data support the hypothesis that immobilization-related degradation of bone mechanical properties is associated with increasing brittleness of cortical bone tissue.

Thermal stabilization of collagen molecules in bone tissue.

Differential thermal calorimetry (DSC) analysis of partially dehydrated bovine bone, demineralized bone and bovine tendon collagen was performed up to 300 degrees C to determine factors influencing stability of mineralized collagen in bone tissue. Two endothermal regions were recognized. The first, attributed to denaturation of collagen triple helix, was hydration dependent and had a peak at 155-165 degrees C in bone, 118-137 degrees C in tendon and 131-136 degrees C in demineralized bone. The second region extended from 245 to 290 degrees C in bone and from 200 to 280 degrees C in tendon and was connected with melting and decomposition of collagen. Differences in thermodynamic parameters between cortical and trabecular bone tissue were stated. Activation energy of collagen unfolding in native bone tissue increased with dehydration of the bone. From the results of the present study we conclude that dehydrated bone collagen is thermally very stable both in native and in demineralized bone. Presence of mineral additionally stabilizes bone tissue.

[Changes in bone strength during convalescence after immobilization induces bone loss--experiment with adult rats]. / Zmiany wytrzymalosci mechanicznej kosci w okresie rekonwalescencji po unieruchomieniu w badaniach na doroslych szczurach.

The aim of the present study was to evaluate the impact of a hindlimb immobilization on mechanical properties of mature bone, both cancellous and cortical tissue. The effectiveness of bone recovery during free activity after remobilization was also studied. The right hindlimb of mature 24 weeks old male rats was immobilized for three weeks. Femora and proximal tibiae methaphyses as well as the straight muscle of thigh were investigated post mortem just after immobilization period or four weeks after remobilization. Mass of the muscle, mass, density and mineralization of bones as well as mechanical properties in axial compression (tibiae metaphyses) or in three-point bending (femora) were measured. After immobilization all parameters were significantly lower in immobilized tibia than in the left, loaded and in respect to the right tibia in the control group. In femur a decrease of dry mass was observed, without any change of breaking force and work to fracture. After four weeks of convalescence, a significant worsening of mechanical competence of femur, and no recovery in tibia was stated. The results obtained indicate that a restoration of mobility without any additional rehabilitation is not enough to stop the processes of bone deterioration induced by unloading in mature bone.

A study of mineral phase in immobilized rat femur: structure refinements by Rietveld analysis.

The aim of the present work was to examine whether immobilization of a limb influences the structure of bone mineral. The mineral phase in rat femora immobilized for 2 weeks during growth was investigated. Bone mineral was subjected to powder X-ray diffraction, using a scanning method after ashing the bones at 630 degrees C. Occupancy factors of ion positions in bone hydroxyapatite (HAP) were analyzed using the Rietveld refinement method. Occupancy factors of the positions OH(-), Ca(2+), and P(-) were significantly lower in immobilized than in control bones, although the position of ions in the HAP structure did not change. Mineralization of tissue in the immobilized bones was lower than in the controls, but there was no correlation between mineralization and occupancy factors. HAP lattice constants in immobilized bones were slightly but significantly different from those in controls. We conclude that the structure of HAP synthesized in bone during temporary lack of loading differs from that of HAP growing under physiological conditions. The Rietveld refinement method proved to be useful in the estimation of the changes in bone mineral.

Age-dependent effect of limb immobilization and remobilization on rat bone.

The effect of unilateral hindlimb immobilization and subsequent free remobilization on bone tissue in rats was examined. Right hindlimb of intensively growing (G), young adult (Y) and adult (A) male rats was immobilized by taping for two weeks. Bone tissue was investigated post mortem in experimental and age-matched control rats, either directly after immobilization (Imm) or after two or four weeks of remobilization (Re2, Re4). Apparent density (d(app)) and mineralization (Min) were estimated in femora and pelvis. The mechanical state of bone tissue in femora was evaluated using an ultrasonic method. Additionally, activity of serum alkaline phosphatase, and serum calcium and phosphorus were measured in each group. Min and d(app) in Imm bones were changed in G rats, while in Y and A only d(app) in Imm femora was affected. Velocity of ultrasound was significantly lower in immobilized femora in each age group, indicating decreased elasticity of bone tissue. The differences between immobilized and control limbs were still significant in Re2 and Re4 groups in G rats. In Y rats the differences between experimental and control bones increased during remobilization. It is concluded that deterioration of bone initiated during two weeks of unloading, last within at least four weeks of free remobilization, despite restoration of normal activity.