Hanna Trębacz

Disuse-induced deterioration of bone strength is not stopped after free remobilization in young adult rats

The effect of unilateral hindlimb immobilization and subsequent free remobilization on mechanical properties of femur was examined in young adult rats. Right hindlimb of 17 weeks old male rats was immobilized for 2 weeks. Rats were sacrificed either directly after immobilization (E0) or after 4 weeks of free remobilization (E4). Mechanical properties in three-point bending as well as dry mass (m(dry)), geometry, apparent density (d(app)), and mineralization of dry bone tissue were measured post mortem in right and left femora of experimental rats (E0, E4) and in right femora of age-matched controls (C0, C4). Differences between right femora of experimental and control animals and between right and left femora of experimental animals were analyzed. After immobilization only d(app) in E0 was significantly lower than in C0. Side-to-side differences in E0 were present only in m(dry) and d(app). Surprisingly, 4 weeks after remobilization the differences between experimental and control femora were more pronounced. Mineralization, d(app), maximum bending moment (M(max)), yield bending moment (M(y)) and stiffness of the right femur were lower in E4 than in age-matched C4. Side-to-side differences in remobilized rats (E4) were still significant for m(dry) and d(app). Additionally, the medullary area was larger, and M(max), M(y), stiffness and work to failure were lower in the right femur than in the left. It is concluded, that the processes of bone deterioration initiated during immobilization do not cease immediately after resumption of normal mechanical loading.

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

Abstract. 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°C. Occupancy factors of ion positions in bone hydroxyapatite (HAP) were analyzed using the Rietveld refinement method. Occupancy factors of the positions OH−, Ca2+, 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.

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.

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

BackgroundCarbon 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.MethodsTwenty-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.ResultsIn 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.ConclusionsDifferences 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.

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 The present paper is focused on the 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.

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.

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.

DSC, FT-IR, NIR, NIR-PCA and NIR-ANOVA for determination of chemical stability of diuretic drugs: impact of excipients

Abstract It is well known that drugs can directly react with excipients. In addition, excipients can be a source of impurities that either directly react with drugs or catalyze their degradation. Thus, binary mixtures of three diuretics, torasemide, furosemide and amiloride with different excipients, i.e. citric acid anhydrous, povidone K25 (PVP), magnesium stearate (Mg stearate), lactose, D-mannitol, glycine, calcium hydrogen phosphate anhydrous (CaHPO4) and starch, were examined to detect interactions. High temperature and humidity or UV/VIS irradiation were applied as stressing conditions. Differential scanning calorimetry (DSC), FT-IR and NIR were used to adequately collect information. In addition, chemometric assessments of NIR signals with principal component analysis (PCA) and ANOVA were applied. Between the excipients examined, lactose and starch did not show any interactions while citric acid, PVP, Mg stearate and glycine were peculiarly operative. Some of these interactions were shown without any stress, while others were caused or accelerated by high temperature and humidity, and less by UV/VIS light. Based on these results, potential mechanisms for the observed interactions were proposed Finally, we conclude that selection of appropriate excipients for torasemide, furosemide and amiloride is an important question to minimize their degradation processes, especially when new types of formulations are being manufactured. Graphical Abstract

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.