Mechanical analysis of a rodent segmental bone defect model: The effects of internal fixation and implant stiffness on load transfer

Segmental bone defect animal models are often used for evaluating the bone regeneration performance of bone substituting biomaterials. Since bone regeneration is dependent on mechanical loading, it is important to determine mechanical load transfer after stabilization of the defect and to study the effects of biomaterial stiffness on the transmitted load. In this study, we assess the mechanical load transmitted over a 6 mm femur defect that is stabilized with an internal PEEK fixation plate. Subsequently, three types of selective laser melted porous titanium implants with different stiffness values were used to graft the defect (five specimens per group). In one additional group, the defect was left empty. Micro strain gauges were used to measure strain values at four different locations of the fixation plate during external loading on the femoral head. The load sharing between the fixation plate and titanium implant was highly variable with standard deviations of measured strain values between 31 and 93% of the mean values. As a consequence, no significant differences were measured between the forces transmitted through the titanium implants with different elastic moduli. Only some non-significant trends were observed in the mean strain values that, consistent with the results of a previous finite element study, implied the force transmitted through the implant increases with the implant stiffness. The applied internal fixation method does not standardize mechanical loading over the defect to enable detecting small differences in bone regeneration performances of bone substituting biomaterials. In conclusion, the fixation method requires further optimization to reduce the effects of the operative procedure and make the mechanical loading more consistent and improve the overall sensitivity of this rat femur defect model.     

The effect of temperature on capacitance changes in an oscillating model membrane

The electrical properties of model membranes are altered during stretching or pressure pulses. We have used a mechanico-electric transduction model to interpret the temperature dependence of capacitance changes produced in oxidized cholesterol membranes during mechanical oscillation. The relative contribution of the torus and bilayer portions of the membrane to the capacitance change is identified. The difference in elasticity between the bilayer and torus decreases rapidly with decreasing temperature and ultimately the torus becomes as solid as the bilayer portion of the model membrane.     

The measurement of shear modulus and membrane surface viscosity of RBC membrane with Ektacytometry: a new technique

A new technique is proposed to estimate the shear modulus (mu) and membrane surface viscosity (eta(m)) of red blood cell (RBC). Theoretical formulae for finding these two parameters are first derived based on the force balance on a RBC in a flow field of low viscosity. Different types of Ektacytometry are then used to measure relevant quantities. The obtained values (mu=6.1 x 10(-6)N/m, eta(m)=8.8 x10 (-7)Ns/m for normal RBC) are consistent with those previously found by micropipette technique and in AC electric field. The present technique is, however, much easier to operate and more advantageous in reflecting the average properties of a large quantity of RBCs, and it is much cheaper to be applied in clinical practice than any other method of measuring the two parameters. The sensitivity of the technique is demonstrated by testing RBCs treated with glutaraldehyde of different concentrations. This technique was demonstrated by the flow chamber.     

Isolation of a rat liver plasma membrane fraction of probable canalicular origin Preparative technique,enzymatic profile,composition, and solute transport

A technique currently used for isolation of brush border membranes from renal and intestinal epithelium that involves vigorous tissue homogenization and sedimentation of non-luminal membranes in the presence of Mg²⁺ has been adapted to rat liver. Liver plasma membranes so prepared consisted almost exclusively of vesicles by electron microscopy, showed some contamination with endoplasmic reticulum and minimal contamination with mitochondria or Golgi by marker enzymes, were highly enriched in alkaline phosphatase, Mg²⁺-ATPase, and 5′-nucleotidase activity compared with homogenate, and showed little enrichment in (Na⁺,K⁺)-ATPase. Comparison of this enzymatic profile with cytochemical studies localizing (Na⁺,K⁺)-ATPase and alkaline phosphatase to the sinusoidal/lateral and canalicular membranes, respectively, suggested that these membranes were predominantly of canalicular origin. They had a lower ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ specific activity, lower lipid content, and higher cholesterol to phospholipid molar ratio than a conventional plasma membrane preparation believed to be enriched in canaliculi. Moreover, it was possible to measure movement of d-[³H]glucose into an osmotically sensitive space bounded by these membrane vesicles.     

The effect of amine structure on complexation with lasalocid in model membrane systems: I. Identification of charged complexes in lipid bilayer membranes

The electrical properties of X-537A (lasalocid) doped lipid bilayer membranes were studied in the presence of a series of nine biogenic amines which contain β-phenylethylamine as the basic structural unit. The ionophore antibiotic was found to form charged complexes within the membrane during the transport of some of the amines. The dependence of membrane conductance on the concentration of ionophore and amine was studied. The amines are divided into three classes according to the nature of the complexes formed: (1) charged complex involving two ionophores (phenylephrine, metanephrine, and amphetamine); (2) charged complex containing three ionophores (dopamine, norepinephrine and epinephrine); and (3) no charged species formed (p- and m-tyramine and β-phenylethylamine).     

Alteration of phospholipid composition of mouse liver microsomes in vivo and the effect on membrane properties

Administration of the methylation inhibitor periodate-oxidized adenosine to male Swiss-Webster mice on a choline-deficient diet produced a decrease (17%) in phosphatidylcholine to phosphatidylethanolamine ratios compared to saline-injected controls in liver, and also in kidney (11%), but not in muscle microsome preparations. Both intact liver microsomes and reconstituted membranes from lipid extracts showed a higher fluorescence anisotropy of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene than control samples in the temperature range of 20–31°C.     

Variations in carotenoid content and acyl chain composition in exponential,stationary and biofilm states of Staphylococcus aureus,and their influence on membrane biophysical properties

Bacteria are often found in close association with surfaces, resulting in the formation of biofilms. In Staphylococcus aureus (S. aureus), biofilms are implicated in the resilience of chronic infections, presenting a serious clinical problem world-wide. Here, S. aureus biofilms are grown under flow within clinical catheters at 37 °C. The lipid composition and biophysical properties of lipid extracts from these biofilms are compared with those from exponential growth and stationary phase cells. Biofilms show a reduction in iso and anteiso branching compensated by an increase in saturated fatty acids compared to stationary phase. A drastic reduction in carotenoid levels is also observed during biofilm formation. Thermotropic measurements of Laurdan GP and DPH polarization, show a reduction of lipid packing at 37 °C for biofilms compared to stationary phase. We studied the effects of carotenoid content on DMPG and DPPG model membranes showing trends in thermotropic behavior consistent with those observed in bacterial isolates, indicating that carotenoids participate in modulating lipid packing. Additionally, bending elastic constant (k_c) measurements using vesicle fluctuation analysis (VFA) show that the presence of carotenoids can increase membrane bending rigidity. The antimicrobial peptide Magainin H2 was less activity on liposomes composed of stationary phase compared to biofilms or exponential growth isolates. This study contributes to an understanding of how Staphylococcus aureus modulates the composition of its membrane lipids, and how those changes affect the biophysical properties of membranes, which in turn may play a role in its virulence and its resistance to different membrane-active antimicrobial agents.     

PEG-amino acid-przewaquinone a conjugations: Synthesis,physicochemical properties and protective effect in a rat model of brain ischemia-reperfusion

A total of 21 PEG-przewaquinone A conjugations with high drug loading ability, good water solubility and in vivo slow-release quality were obtained by conjugating przewaquinone A with PEG through amino acids and tripeptides spacers respectively. Notably, compound 3a can obviously reduce the brain ischemia-reperfusion damage dose-dependently in a rat model, which indicated the efficacy of our PEG prodrug strategy.     

The peritrophic membrane: Ultrastructural analysis and function as a mechanical barrier to microbial infection in Orgyia pseudotsugata

The feasibility of freeze-etching as a method for structural analysis of the peritrophic membrane (PM) has been demonstrated, and this method and other electron microscopical techniques have been employed to investigate the possible function of the PM as a mechanical barrier to infection of the larval midgut. Thin sections show that the PM consists of granular and microfibrous layers typical of PMs from other insects. Freeze-etch studies show that the membrane consists of at least three structurally distinct layers. The major component is a layer of chitin microfibrils that appear to be arranged in oriented layers. The fibrils exhibit a periodicity of 4 nm and are embedded in a granular matrix. The fibrils coalesce into 100-nm fibers which run throughout the microfibrillar layer. Adjacent to this layer is a very rough layer, with randomly dispersed granules 100 nm in diameter and covered with particles 32 nm in diameter. Adjacent to the midgut lumen is a finely granular layer which gives the PM a smooth appearance. No pores or other discontinuities through which a bacterial or viral pathogen could penetrate were detected in the PM, indicating the PM of the Douglas fir tussock moth could function as a mechanical barrier to microbial infection. The importance of chitin and protein to the apparent strength of the Douglas fir tussock moth PM was partially elucidated by studies with chitinases and proteases. Both types of enzymes degraded the PM, causing release of the products of hyrolysis or structural changes in the membrane.     

The mechanical environment of the chondrocyte: a biphasic finite element model of cell-matrix interactions in articular cartilage

Mechanical compression of the cartilage extracellular matrix has a significant effect on the metabolic activity of the chondrocytes. However, the relationship between the stress–strain and fluid-flow fields at the macroscopic “tissue” level and those at the microscopic “cellular” level are not fully understood. Based on the existing experimental data on the deformation behavior and biomechanical properties of articular cartilage and chondrocytes, a multi-scale biphasic finite element model was developed of the chondrocyte as a spheroidal inclusion embedded within the extracellular matrix of a cartilage explant. The mechanical environment at the cellular level was found to be time-varying and inhomogeneous, and the large difference (3 orders of magnitude) in the elastic properties of the chondrocyte and those of the extracellular matrix results in stress concentrations at the cell–matrix border and a nearly two-fold increase in strain and dilatation (volume change) at the cellular level, as compared to the macroscopic level. The presence of a narrow “pericellular matrix” with different properties than that of the chondrocyte or extracellular matrix significantly altered the principal stress and strain magnitudes within the chondrocyte, suggesting a functional biomechanical role for the pericellular matrix. These findings suggest that even under simple compressive loading conditions, chondrocytes are subjected to a complex local mechanical environment consisting of tension, compression, shear, and fluid pressure. Knowledge of the local stress and strain fields in the extracellular matrix is an important step in the interpretation of studies of mechanical signal transduction in cartilage explant culture models.     

The effect of assay composition, detergent solubilization and reconstitution on red beet (Beta vulgaris L.) plasma membrane H-ATPase kinetic properties

Modification of the salt concentration, composition and/or buffer type in the assay of plasma membrane ATPase activity caused substantial changes in the K_m and slight changes in the temperature dependence of this enzyme. The K_m and temperature dependence were also affected by detergent solubilization of the ATPase and its subsequent reconstitution into liposomes. Modulation of kinetic properties by assay composition and hydrophobic state reflect the sensitivity of the plasma membrane H⁺-ATPase to its immediate environment. This may indicate a possible regulatory mechanism for this important plant enzyme.     

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or chaning the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5′-(N-ethylcar☐amido)adenosine (NECA, 10 μM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 ± 0.02 (S.E.) to 1.80 ± 0.02 (p < 0.001) while inosine (10 μM), a poor adenosine receptor agonist, had no effect (1.73 ± 0.04, p =n.s. vs. control, p < 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA > adenosine = N⁶-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 μM) did not significantly change the number or affinity of [³H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Feasibility,effectiveness, and safety of a novel cryo-balloon targeted lung denervation technique in an animal model

Background and objectiveTargeted lung denervation (TLD) is a pulmonary interventional procedure for COPD that aims to disrupt parasympathetic nerve input to the lung to reduce the clinical consequences of cholinergic hyperactivity. TLD has been proven to be a safe procedure and effectively alleviate symptoms and reduce the onset of exacerbation. In the present study, we developed a novel cryo-balloon TLD system and evaluated its feasibility, safety, and effectiveness.MethodsA preclinical study was performed on twelve sheep, four were tested for airway resistance alterations before and after TLD, two were tested for the Hering-Breuer reflex (HBR) and the remaining six sheep were evaluated for 28 days to assess the safety and effectiveness of the procedure.ResultsAfter an observation period of 28 days, significant disruption of vagal innervation to the lung could be validated by both histological and physiological assessments. The operation time was shorter than traditional procedure, with minimal adjacent tissue injury and no device-related adverse events.ConclusionsThe novel cryo-balloon TLD procedure was feasible, safe, and effective. In comparison with the traditional procedure, this treatment system required shorter operation time and caused less denervation-induced damage to adjacent tissues.     

To form and function: on the role of basement membrane mechanics in tissue development,homeostasis and disease

The basement membrane (BM) is a special type of extracellular matrix that lines the basal side of epithelial and endothelial tissues. Functionally, the BM is important for providing physical and biochemical cues to the overlying cells, sculpting the tissue into its correct size and shape. In this review, we focus on recent studies that have unveiled the complex mechanical properties of the BM. We discuss how these properties can change during development, homeostasis and disease via different molecular mechanisms, and the subsequent impact on tissue form and function in a variety of organisms. We also explore how better characterization of BM mechanics can contribute to disease diagnosis and treatment, as well as development of better in silico and in vitro models that not only impact the fields of tissue engineering and regenerative medicine, but can also reduce the use of animals in research.     

Structural features of model glycopeptides in solution and in membrane phase: a spectroscopic and molecular mechanics investigation

Model glycopeptides of the general formula Boc-Ala-Thr(G-D)-A(1)-A(2)-Leu-Leu-Lys(N)-Ala-OMe, where D = dansyl (dimethyl aminonaphthalenesulphonyl), G = glucosyl and N = naphthyl, while A(1)-A(2) = Ala-Leu or Aib-Aib, and denoted as D-G-Ala-N and D-G-Aib-N, respectively, were used to investigate glycoprotein-membrane interactions. They carry two fluorophores (D and N), covalently linked to the glucose ring and the lysine side chain, respectively, while the threonine side chain is O-glycosylated. CD spectra in different solvent media suggest that both glycopeptides attain an ordered structure, possibly a helix-like conformation. By combining FRET (fluorescence resonance energy transfer) experiments with molecular mechanics data, the most probable structures of both glycopeptides were built up, starting from both a right-handed (rh) alpha- and 3(10)-helix. They were found to populate an alpha-helical conformation, a result further confirmed by the very good agreement between theoretical and experimental quenching efficiency only observed when the backbone chain was in alpha-helix. The association of D-G-Ala-N with model membranes (liposomes) was studied by CD, fluorescence decay, fluorescence anisotropy, and collisional quenching experiments. The binding does not alter the structural features of the peptide because the CD spectral patterns are unaffected by the association. The peptide orientation inside the phospholipidic bilayer is guided by the polar glucose molecule lying in the water phase. The insertion of the hydrophobic backbone chain into the membrane, seeing the probes only partially accessible from the external solution, is characterized by a significant degree of heterogeneity, an increase in vesicles size, and a relevant stabilizing effect on the membrane itself against rupture by methanol.     

The effect of thioglycolate on intermediate filaments and membrane translocation in rat urothelium during the expansion-contraction cycle

Summary The functional role of cytokeratin intermediate filaments in the translocation of asymmetric membrane plaques between cytoplasm and surface of apical urothelial cells was investigated during contraction and expansion of rat urinary bladders. A stereological investigation of electron micrographs provided estimations of surface area, volume, and number of discoidal vesicles and infoldings per unit volume of urothelial apical cell cytoplasm. Contracted and distended bladders incubated in 0.01 M sodium bicarbonate were compared to identical preparations experimentally incubated in 5 mM thioglycolic acid. The latter reagent disrupts the intermediate filament network by reducing sulfhydryl bridges. Densities of discoidal vesicles in cells contracted after incubation in thioglycolate were similar to density estimations in cells expanded under control conditions. Similarly, densities of vesicles in cells expanded after exposure to thioglycolate were comparable in number to those in normally contracted cells. Thus, membrane translocation to and from the luminal surface was blocked by thioglycolate treatment. The lack of normal membrane transfer at the luminal surface induces apical cells exposed to experimental conditions to undergo extraordinary adjustments in response to external pressures of bladder contraction and distension. During contraction, the apical-intermediate cell interface unfolded while the luminal surface ballooned out into the lumen. In distended bladders, large intercellular spaces formed between apical cells along their lateral margins. The results support a model published earlier implicating the filament network as a critical mediator of membrane translocation.     

Mechanokinetic model of cell membrane: theoretical analysis of plasmalemma homeostasis, growth and division

A theoretical model dealing with endocytosis, exocytosis and caveolae invagination, describing plasmalemma homeostasis during cell growth and division, is proposed. It considers transmembrane pressure, membrane tension and mechanosensitivity of membrane processes. Membrane hydraulic conductivity and the flux of transmembrane nonvesicular transport are taken into account. The developed mathematical analysis operates with a formulated set of constitutive equations describing the mechanical state and kinetics of changes in an open dynamic membrane system. The standard version of a model with adjusted parameters was implemented, and predictions including a discussion on the effect of possible parameter modifications were presented. Computer simulations indicate big changes in the magnitude of membrane tension and elasticity, and in the number of membrane buddings in young cells and during mitosis. They also show the extent of cell growth inhibition resulting from a decrease in transmembrane transport or an increase in the exerted difference in osmotic pressure. Moreover, the simulations reveal that exocytosis regulated during mitosis may not be as important for cell growth, as sometimes presumed. Finally, practical application and possible extension of the model are discussed.     

益生菌对肠易激综合征小鼠肠道黏膜CRF-R1表达及肠道屏障功能的影响

目的通过观察肠易激综合征(IBS)小鼠模型结肠黏膜肥大细胞上CRF-R1及结肠黏膜PAR-2、Claudin1~4等的表达变化,探讨IBS中应激通过肥大细胞引起肠道屏障功能障碍的可能机制并观察婴儿双歧杆菌的治疗作用。方法 30只雄性Balb/c小鼠随机分为对照组、模型组及婴儿双歧杆菌组。以束缚应激法建立IBS小鼠模型。婴儿双歧杆菌组给予婴儿双歧杆菌灌胃,而对照组及模型组给予等体积生理盐水灌胃。观测腹肌收缩反射(AWR)后处死小鼠。ELISA检测外周血中类胰蛋白酶的表达变化。免疫组织化学分析结肠黏膜CRF、PAR-2、Claudin1、Claudin2、Claudin3、Claudin4的表达情况。免疫荧光双标分析结肠黏膜CRF-R1在肥大细胞的表达情况。RT-PCR检测结肠CRF-R1mRNA的表达情况。结果与对照组相比,模型组小鼠外周血类胰蛋白酶表达量增加;结肠黏膜中CRF、PAR-2、Claudin2表达量、CRF-R1+肥大细胞数目及CRF-R1 mRNA表达量增加,结肠黏膜Claudin1、Claudin3、Claudin4表达量降低,差异均有统计学意义(P0.05)。婴儿双歧杆菌干预后,该组小鼠外周血中类胰蛋白酶表达量降低;结肠黏膜中CRF、PAR-2、Claudin2表达量、CRF-R1+肥大细胞数目及CRF-R1 mRNA表达量降低,结肠黏膜Claudin1、Claudin3、Claudin4表达量增高,差异均有统计学意义(P0.05)。结论 IBS小鼠模型中,婴儿双歧杆菌可以减轻肠道屏障功能障碍,其机制可能与抑制结肠黏膜肥大细胞上CRF-R1的表达而抑制肥大细胞的激活及其免疫因子的释放,从而降低结肠黏膜PAR-2的表达有关。     

Gamma-linolenic acid alters the composition of mitochondrial membrane subfractions,decreases outer mitochondrial membrane binding of hexokinase and alters carnitine palmitoyltransferase I properties in the Walker 256 rat tumour

Gamma-linolenic acid (GLA) is known to be an inhibitor of Walker 256 tumour growth in vivo and causes changes in both mitochondrial structure and cellular metabolism. The aim of the present study was to investigate in greater detail the changes in energy metabolism and ultrastructure induced by GLA in this tumour model. A diet containing 5.5% GLA, which is sufficient to cause a 45% decrease in tumour growth, was found to almost double the triacylglycerol (TAG) content of the tumour and to increase the quantity of 20:3 n?6, 20:4 n?6, 22:4 n?6 and 22:5 n?6 in the TAG fraction as determined by gas chromatography–mass spectrometry (GCMS) analysis. Morphometric analysis of the tumour by electron microscopy confirmed this increase in TAG content, identifying a doubling of lipid droplet content in the GLA dietary group. The surface density of mitochondrial cristae was reduced, along with a reduction in the number of contact sites (CS) and matrix granules. These three parameters are likely indicators of a reduction in mitochondrial metabolic activity. Measurement of hexokinase activity identified that much of the total hexokinase activity was in the mitochondrially bound form (66.5%) in the control tumour and that GLA caused a decrease in the amount of enzyme in the bound form (39.3%). The fatty acyl chain composition of the tumour mitochondrial subfractions, outer membranes (OM), CSs and inner membranes (IM) was determined by GCMS. All subfractions showed considerable increases in 20:3 n?6 and decreases in 18:1 n?9, 18:2 n?6 and 22:6 n?3, when exposed to GLA diet. These changes were reflected in a large increase in the n?6/n?3 ratio in the GLA OM vs. the control OM, 21.299 vs. 6.747, respectively. The maximal activity of OM carnitine palmitoyltransferase I (CPT I) was found to be decreased by 61.6% in the GLA diet group. This was accompanied by a decrease in malonyl CoA sensitivity and a decrease in affinity for 16:0 CoA substrate. Such changes in CPT I may be the cause of cytoplasmic acyl CoA accumulation seen in this tumour model. These effects, together with previously reported increases in lipid peroxidation, lead to the conclusion that GLA may cause inhibition of tumour cell growth through separate but interlinked pathways, all of which eventually lead to apoptosis and a decrease in tumour development. The influence of mitochondrial OM fatty acyl chain composition upon two important enzymes of energy metabolism, hexokinase and CPT I, both of which have been linked to apoptosis, is of considerable importance for future studies on fatty acid-induced cell death.