Biomechanical properties of murine TMJ articular disc and condyle cartilage via AFM-nanoindentation

This study aims to quantify the biomechanical properties of murine temporomandibular joint (TMJ) articular disc and condyle cartilage using AFM-nanoindentation. For skeletally mature, 3-month old mice, the surface of condyle cartilage was found to be significantly stiffer (306 ± 84 kPa, mean ± 95% CI) than those of the superior (85 ± 23 kPa) and inferior (45 ± 12 kPa) sides of the articular disc. On the disc surface, significant heterogeneity was also detected across multiple anatomical sites, with the posterior end being the stiffest and central region being the softest. Using SEM, this study also found that the surfaces of disc are composed of anteroposteriorly oriented collagen fibers, which are sporadically covered by thinner random fibrils. Such fibrous nature results in both an F-D^3/2 indentation response, which is a typical Hertzian response for soft continuum tissue under a spherical tip, and a linear F-D response, which is typical for fibrous tissues, further signifying the high degree of tissue heterogeneity. In comparison, the surface of condyle cartilage is dominated by thinner, randomly oriented collagen fibrils, leading to Hertzian-dominated indentation responses. As the first biomechanical study of murine TMJ, this work will provide a basis for future investigations of TMJ tissue development and osteoarthritis in various murine TMJ models.     

Methane output of rabbits (Oryctolagus cuniculus) and guinea pigs (Cavia porcellus) fed a hay-only diet: implications for the scaling of methane production with body mass in non-ruminant mammalian herbivores

It is assumed that small herbivores produce negligible amounts of methane, but it is unclear whether this is a physiological peculiarity or simply a scaling effect. A respiratory chamber experiment was conducted with six rabbits (Oryctolagus cuniculus, 1.57 ± 0.31 kg body mass) and six guinea pigs (Cavia porcellus, 0.79 ± 0.07 kg) offered grass hay ad libitum. Daily dry matter (DM) intake and DM digestibility were 50 ± 6 g kg^? 0.75 d^? 1 and 55 ± 6% in rabbits and 59 ± 11 g kg^? 0.75 d^? 1 and 61 ± 3% in guinea pigs, respectively. Methane production was similar for both species (0.20 ± 0.10 L d^? 1 and 0.22 ± 0.08 L d^? 1) and represented 0.69 ± 0.32 and 1.03 ± 0.29% of gross energy intake in rabbits and guinea pigs, respectively. In relation to body mass (BM) guinea pigs produced significantly more methane. The data on methane per unit of BM obtained in this study and from the literature on the methane output of elephant, wallabies and hyraxes all lay close to a regression line derived from roughage-fed horses, showing an increase in methane output with BM. The regression, including all data, was nearly identical to that based on the horse data only (methane production in horses [L d^? 1] = 0.18 BM [kg]^{0.97 (95%CI 0.92–1.02)}) and indicates linear scaling. Because feed intake typically scales to BM^0.75, linear scaling of methane output translates into increasing energetic losses at increasing BM. Accordingly, the data collection indicates that an increasing proportion of ingested gross energy is lost because relative methane production increases with BM. Different from ruminants, such losses (1%–2% of gross energy) appear too small in non-ruminant herbivores to represent a physiologic constraint on body size. Nevertheless, this relationship may represent a physiological disadvantage with increasing herbivore body size.     

Seed rain and seed bank of Chinese yew (Taxus chinensis var. mairei) population in Tianmu Mountain☆

Seed rain and seed bank of a Chinese yew (Taxus chinensis var. mairei) population in Tianmu Mountain were researched in 2008 and 2009. The seed rain lasted from 16th–23th of October to 5th–14th of December, and the heaviest seed falling period was from 2nd to18th of November. The intensity of seed rain showed a great inter-annual variation, with a good harvest in 2008. The fallen seeds were composed of 49.9% proportion of immature seed, 33.8% proportion of chewed seed and 16.3% proportion of mature seed. The analysis on the soil seed bank under mother forest showed that the number of intact seeds was 122.75 ± 108.08 grain/m² in October, 279.25 ± 210.73 grain/m² in December 2008, and 166.5 ± 165.34 grain/m² in October, 322.5 ± 275.73 grain/m² in December 2009. The increased number of seed was 156.5 ± 222.723 grain/m² in 2008 and 156 ± 275grain/m² in 2009, which showed a significant variation. Large number of intact seeds added into soil seed bank after seed rain each year. The number of intact seeds in soil seed bank decreased 112.75 ± 47.74 grain/m² from December 2008 to October 2009. Large number of intact seeds lost from seed rot and seed predation by animals. The number of seeds in soil bank under bamboo forest was much lower than that of mother tree forest, and the increased number of seeds was 0.63 ± 1.60 grain/m² in 2008 and 2.88 ± 1.86 grain/m² in 2009. The number of seedling was 0.73 ± 1.10 trees/m² in mother tree forest and 0.09 ± 0.35 trees/m² in bamboo forest. Seedling survival ratio was 0.37% in mother tree forest and 10.23% in bamboo forest. The micro-habitat in bamboo forest was fit for seed germination. Birds transported seeds to bamboo forest, and had an important effect on the regeneration of Chinese yew.     

The region-dependent biphasic viscoelastic properties of human temporomandibular joint discs under confined compression

The objective of this study was to determine the biphasic viscoelastic properties of human temporomandibular joint (TMJ) discs, correlate these properties with disc biochemical composition, and examine the relationship between these properties and disc dynamic behavior in confined compression. The equilibrium aggregate modulus (H_A), hydraulic permeability (k), and dynamic modulus were examined between five disc regions. Biochemical assays were conducted to quantify the amount of water, collagen, and glycosaminoglycan (GAG) content in each region. The creep tests showed that the average equilibrium moduli of the intermediate, lateral, and medial regions were significantly higher than for the anterior and posterior regions (69.75±11.47 kPa compared to 22.0±5.15 kPa). Permeability showed the inverse trend with the largest values in the anterior and posterior regions (8.51±1.36×10^?15 m⁴/Ns compared with 3.75±0.72×10^?15 m⁴/Ns). Discs were 74.5% water by wet weight, 62% collagen, and 3.2% GAG by dry weight. Regional variations were only observed for water content which likely results in the regional variation in biphasic mechanical properties. The dynamic modulus of samples during confined compression is related to the aggregate modulus and hydraulic permeability of the tissue. The anterior and posterior regions displayed lower complex moduli over all frequencies (0.01–3 Hz) with average moduli of 171.8–609.3 kPa compared with 454.6–1613.0 kPa for the 3 central regions. The region of the TMJ disc with higher aggregate modulus and lower permeability had higher dynamic modulus. Our results suggested that fluid pressurization plays a significant role in the load support of the TMJ disc under dynamic loading conditions.     

Cryopreserved mobilized autologous blood progenitors stored for more than 2 years successfully support blood count recovery after high-dose chemotherapy

Background aimsThe ability of hematopoietic progenitor cells–apheresis (HPC-A) that have been stored for many years after cryopreservation to reconstitute hematopoiesis following high-dose chemo/radiotherapy has not been well-documented.MethodsIn this retrospective study, eight Canadian centers contributed data from 53 autologous stem cell transplants (ASCT) performed using HPC-A that had undergone long-term storage (>2 years, range 2–7 years) and 120 ASCT using HPC-A stored for <6 months (short-term storage).ResultsThe doses of nucleated and CD34⁺ cells per kilogram recipient weight were similar between the short- (mean ± SD, 4.7 ± 4.9 × 10⁸ and 6.8 ± 4.3 × 10⁶, respectively) and long- (4.0 ± 4.9 × 10⁸ and 6.1 ± 3.4 × 10⁶, respectively) term storage groups. The median days to neutrophils (absolute neutrophil count; ANC) >0.5 × 10⁹/L (median 11 days for both short- and long-term storage) and platelets >20 × 10⁹/L (median 12 and 11 for short- and long-term storage, respectively) post-ASCT were not significantly different between the two groups. When ASCT performed with <5 × 10⁶/kg CD34⁺ cells was compared there was also no difference in ANC or platelet recovery (median 12 days for both after short-term storage, and 12 and 11 days, respectively, after long-term storage). Fourteen HPC-A products stored for >5 years also showed similar count recoveries as the entire long-term storage group (median 11 days for both ANC and platelets).ConclusionsCryopreserved HPC-A can be stored for at least 5 years with no apparent loss in their ability to support hematopoietic reconstitution after high-dose chemotherapy.     

Development and validation of a procedure to isolate viable bone marrow cells from the vertebrae of cadaveric organ donors for composite organ grafting

Background aimsDonor-derived vertebral bone marrow (BM) has been proposed to promote chimerism in solid organ transplantation with cadaveric organs. Reports of successful weaning from immunosuppression in patients receiving directed donor transplants in combination with donor BM or blood cells and novel peri-transplant immunosuppression has renewed interest in implementing similar protocols with cadaveric organs.MethodsWe performed six pre-clinical full-scale separations to adapt vertebral BM preparations to a good manufacturing practice (GMP) environment. Vertebral bodies L4–T8 were transported to a class 10 000 clean room, cleaned of soft tissue, divided and crushed in a prototype bone grinder. Bone fragments were irrigated with medium containing saline, albumin, DNAse and gentamicin, and strained through stainless steel sieves. Additional cells were eluted after two rounds of agitation using a prototype BM tumbler.ResultsThe majority of recovered cells (70.9 ± 14.1%, mean ± SD) were eluted directly from the crushed bone, whereas 22.3% and 5.9% were eluted after the first and second rounds of tumbling, respectively. Cells were pooled and filtered (500, 200 μm) using a BM collection kit. Larger lumbar vertebrae yielded about 1.6 times the cells of thoracic vertebrae. The average product yielded 5.2 ± 1.2 × 10¹⁰ total cells, 6.2 ± 2.2 × 10⁸ of which were CD45⁺ CD34⁺. Viability was 96.6 ± 1.9% and 99.1 ± 0.8%, respectively. Multicolor flow cytometry revealed distinct populations of CD34⁺ CD90⁺ CD117^dim hematopoietic stem cells (15.5 ± 7.5% of the CD34 ⁺ cells) and CD45^? CD73⁺ CD105⁺ mesenchymal stromal cells (0.04 ± 0.04% of the total cells).ConclusionsThis procedure can be used to prepare clinical-grade cells suitable for use in human allotransplantation in a GMP environment.     

Intra-operative preparation of autologous bone marrow-derived CD34-enriched cellular products for cardiac therapy

Background and AimsWith the advent of regenerative therapy, there is renewed interest in the use of bone marrow as a source of adult stem and progenitor cells, including cell subsets prepared by immunomagnetic selection. Cell selection must be rapid, efficient and performed according to current good manufacturing practices. In this report we present a methodology for intra-operative preparation of CD34⁺ selected autologous bone marrow for autologous use in patients receiving coronary artery bypass grafts or left ventricular assist devices.Methods and ResultsWe developed a rapid erythrocyte depletion method using hydroxyethyl starch and low-speed centrifugation to prepare large-scale (mean 359 mL) bone marrow aspirates for separation on a Baxter Isolex 300i immunomagnetic cell separation device. CD34 recovery after erythrocyte depletion was 68.3 ± 20.2%, with an average depletion of 91.2 ± 2.8% and an average CD34 content of 0.58 ± 0.27%. After separation, CD34 purity was 64.1 ± 17.2%, with 44.3 ± 26.1% recovery and an average dose of 5.0 ± 2.7 × 10⁶ CD34⁺ cells/product. In uncomplicated cases CD34-enriched cellular products could be accessioned, prepared, tested for release and administered within 6 h. Further analysis of CD34⁺ bone marrow cells revealed a significant proportion of CD45^? CD34⁺ cells.ConclusionsIntra-operative immunomagnetic separation of CD34-enriched bone marrow is feasible using rapid low-speed Hetastarch sedimentation for erythrocyte depletion. The resulting CD34-enriched product contains CD45^? cells that may represent non-hematopoietic or very early hematopoietic stem cells that participate in tissue regeneration.     

Improved survival of mesenchymal stromal cell after hypoxia preconditioning: role of oxidative stress

AimsTo investigate the mechanisms underlying the beneficial effect of hypoxia preconditioning (HPC) on mesenchymal stromal cells (MSCs) and optimize novel non-invasive methods to assess the effect of biological interventions aimed to increased cell survival.Main methodsMSCs from rat femur, with or without HPC, were exposed to hypoxic conditions in cell culture (1% O₂ for 24 h) and cell survival (by the LDH release assay and Annexin-V staining) was measured. Oxidant status (conversion of dichloro-fluorescein-DCF- and dihydro-ethidium-DHE-, protein expression of oxidant enzymes) was characterized, together with the mobility pattern of cells under stress. Furthermore, cell survival was assessed non-invasively using state-of-the-art molecular imaging.Key findingsCompared to controls, Hypoxia resulted in increased expression of the oxidative stress enzyme NAD(P)H oxidase (subunit 67^phox: 0.05 ± 0.01 AU and 0.48 ± 0.02 AU, respectively, p < 0.05) and in the amount of ROS (DCF: 13 ± 1 and 42 ± 3 RFU/μg protein, respectively, p < 0.05) which led to a decrease in stem cell viability. Hypoxia preconditioning preserved cell biology, as evidenced by preservation of oxidant status (16 ± 1 RFU/μg protein, p < 0.05 vs. hypoxia), and cell viability. Most importantly, the beneficial effect of HPC can be assessed non-invasively using molecular imaging.SignificanceHPC preserves cell viability and function, in part through preservation of oxidant status, and its effects can be assessed using state-of-the-art molecular imaging. Understanding of the mechanisms underlying the fate of stem cells will be critical for the advancement of the field of stem cell therapy.     

The kinetics and regulation of phosphofructokinase from Teladorsagia circumcincta

Phosphofructokinase (PFK-1) activity was examined in L₃ and adult Teladorsagia circumcincta, both of which exhibit oxygen consumption. Although activities were higher in the adult stage, the kinetic properties of the enzyme were similar in both life cycle stages. T. circumcincta PFK-1 was subject to allosteric inhibition by high ATP concentration, which increased both the Hill coefficient (from 1.4 ± 0.2 to 1.7 ± 0.2 in L₃s and 2.0 ± 0.3 to 2.4 ± 0.4 in adults) and the K_½ for fructose 6 phosphate (from 0.35 ± 0.02 to 0.75 ± 0.05 mM in L₃s and 0.40 ± 0.03 to 0.65 ± 0.05 mM in adults). The inhibitory effects of high ATP concentration could be reversed by fructose 2,6 bisphosphate and AMP, but glucose 1,6 bisphosphate had no effect on activity. Similarly, phosphoenolpyruvate had no effect on activity, while citrate, isocitrate and malate exerted mild inhibitory effects, but only at concentrations exceeding 2 mM. The observed kinetic properties for T. circumcincta PFK-1 were very similar to those reported for purified Ascaris suum PFK-1, though slight differences in sensitivity to ATP concentration suggests there may be subtle variations at the active site. These results are consistent with the conservation of properties of PFK-1 amongst nematode species, despite between species variation in the ability to utilise oxygen.     

Production of interspecies handmade cloned embryos by nuclear transfer of cattle, goat and rat fibroblasts to buffalo (Bubalus bubalis) oocytes

The possibility of producing interspecies handmade cloned (iHMC) embryos by nuclear transfer from donor cells of cattle, goat and rat using buffalo oocytes as recipient cytoplasts was explored. Zona-free buffalo oocytes were enucleated by protrusion cone-guided bisection with a microblade. After electrofusion with somatic cells, reconstructed oocytes were activated by calcimycin A23187, treated with 6-dimethylaminopurine and were cultured in K-RVCL-50^® medium for 8 days. Although the cleavage rate was not significantly different when buffalo, cattle, goat or rat cells were used as donor nuclei (74.6 ± 3.8, 82.8 ± 5.3, 86.0 ± 4.9 and 82.3 ± 3.6%, respectively), the blastocyst rate was significantly higher (P < 0.01) for buffalo (51.4 ± 2.6) than for cattle (3.5 ± 1.0) or the goat (2.2 ± 0.9), whereas none of the embryos crossed the 32-cell stage when rat cells were used. However, the total cell number was similar for buffalo–buffalo (175.0 ± 5.07) and cattle–buffalo embryos (178.0 ± 11.84). Following transfer of 3 buffalo–buffalo embryos each to 6 recipients, 3 were found to be pregnant, though the pregnancies were not carried to full term. These results suggest that interspecies blastocyst stage embryos can be produced by iHMC using buffalo cytoplasts and differentiated somatic cells from cattle and goat and that the source of donor nucleus affects the developmental competence of interspecies embryos.     

Viscoelastic properties of human cerebellum using magnetic resonance elastography

BackgroundThe cerebellum has never been mechanically characterised, despite its physiological importance in the control of motion and the clinical prevalence of cerebellar pathologies. The aim of this study was to measure the linear viscoelastic properties of the cerebellum in human volunteers using Magnetic Resonance Elastography (MRE).MethodsCoronal plane brain 3D MRE data was performed on eight healthy adult volunteers, at 80 Hz, to compare the properties of cerebral and cerebellar tissues. The linear viscoelastic storage (G′) and loss moduli (G″) were estimated from the MRE wave images by solving the wave equation for propagation through an isotropic linear viscoelastic solid. Contributions of the compressional wave were removed via application of the curl-operator.ResultsThe storage modulus for the cerebellum was found to be significantly lower than that for the cerebrum, for both white and grey matter. Cerebrum: white matter (mean±SD) G′=2.41±0.23 kPa, grey matter G′=2.34±0.22 kPa; cerebellum: white matter, G′=1.85±0.18 kPa, grey matter G′=1.77±0.24 kPa; cerebrum vs cerebellum, p<0.001. For the viscous behaviour, there were differences in between regions and also by tissue type, with the white matter being more viscous than grey matter and the cerebrum more viscous than the cerebellum. Cerebrum: white matter G″=1.21±0.21 kPa, grey matter G″=1.11±0.03 kPa; cerebellum: white matter G″=1.1±0.23 kPa, grey matter G″=0.94±0.17 kPa.DiscussionThese data represent the first available data on the viscoelastic properties of cerebellum, which suggest that the cerebellum is less physically stiff than the cerebrum, possibly leading to a different response to mechanical loading. These data will be useful for modelling of the cerebellum for a range of purposes.     

Action of ANP on the nongenomic dose-dependent biphasic effect of aldosterone on NHE1 in proximal S3 segment

The rapid (2 min) nongenomic effects of aldosterone (ALDO) and/or spironolactone (MR antagonist), RU 486 (GR antagonist), atrial natriuretic peptide (ANP) and dimethyl-BAPTA (BAPTA) on the intracellular pH recovery rate (pHirr) via NHE1 (basolateral Na⁺/H⁺ exchanger isoform), after the acid load induced by NH₄Cl, and on the cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in the proximal S3 segment isolated from rats, by the probes BCECF-AM and FLUO-4-AM, respectively. The basal pHi was 7.15 ± 0.008 and the basal pHirr was 0.195 ± 0.012 pH units/min (number of tubules/number of tubular areas = 16/96). Our results confirmed the rapid biphasic effect of ALDO on NHE1: ALDO (10^?12 M) increases the pHirr to approximately 59% of control value, and ALDO (10^?6 M) decreases it to approximately 49%. Spironolactone did not change these effects, but RU 486 inhibited the stimulatory effect and maintained the inhibitory effect. ANP (10^?6 M) or BAPTA (5 × 10^?5 M) alone had no significant effect on NHE1 but prevented both effects of ALDO on this exchanger. The basal [Ca²⁺]_i was 104 ± 3 nM (15), and ALDO (10^?12 or 10^?6 M) increased the basal [Ca²⁺]_i to approximately 50% or 124%, respectively. RU 486, ANP and BAPTA decreased the [Ca²⁺]_i and inhibited the stimulatory effect of both doses of ALDO. The results suggest the involvement of GR on the nongenomic effects of ALDO and indicate a pHirr-regulating role for [Ca²⁺]_i that is mediated by NHE1, stimulated/impaired by ALDO, and affected by ANP or BAPTA with ALDO. The observed nongenomic hormonal interaction in the S3 segment may represent a rapid and physiologically relevant regulatory mechanism in the intact animal under conditions of volume alterations.     

Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Background aimsMesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues.MethodsMSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive^® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays.ResultsPost-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure.ConclusionsA method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.     

Synthesis of highly potent novel anti-tubercular isoniazid analogues with preliminary pharmacokinetic evaluation

Thirty two novel isoniazid analogues were prepared by one-pot three component condensations of isoniazid (INH), 3-mercaptopropionic acid and various aryl/heteroaryl aldehydes. The synthesized compounds were evaluated for their anti-TB activity against Mycobacterium tuberculosis H37Rv (MTB) and cytotoxicity. Among the compounds, compound N-(2-(4-(benzyloxy) phenyl)-4-oxo-1,3-thiazinan-3-yl) isonicotinamide (17) inhibited MTB with MIC of 0.12 μM and was three times more potent than INH. The main pharmacokinetic parameters after intravenous administration (10 mg/kg body weight) in male Wistar rats viz. t_½, K_el, mean plasma clearance and mean volume of distribution were found to be 1.14 ± 0.20 h, 0.62 ± 0.10 h^?1, 22.48 ± 0.16 mL/kg/min and 1.99 ± 0.49 L, respectively. The systemic absorption was slow after oral administration (50 mg/kg body weight). The peak plasma concentration was found to be 1.31 ± 0.06 μg/mL attained in 3 h. The bioavailability was found to be 16.7%.     

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol–disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV–Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK_a of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 ± 0.2) × 10⁷ M^? 1 s^? 1) and peroxynitrite ((3.7 ± 0.4) × 10⁵ M^? 1 s^? 1) at pH 7.4 and 25 °C.     

Hyperosmolaric contrast agents in cartilage tomography may expose cartilage to overload-induced cell death

In clinical arthrographic examination, strong hypertonic contrast agents are injected directly into the joint space. This may reduce the stiffness of articular cartilage, which is further hypothesized to lead to overload-induced cell death. We investigated the cell death in articular cartilage while the tissue was compressed in situ in physiological saline solution and in full strength hypertonic X-ray contrast agent Hexabrix^TM. Samples were prepared from bovine patellae and stored in Dulbecco’s Modified Eagle’s Medium overnight. Further, impact tests with or without creep were conducted for the samples with contact stresses and creep times changing from 1 MPa to 10 MPa and from 0 min to 15 min, respectively. Finally, depth-dependent cell viability was assessed with a confocal microscope. In order to characterize changes in the biomechanical properties of cartilage as a result of the use of Hexabrix?, stress-relaxation tests were conducted for the samples immersed in Hexabrix? and phosphate buffered saline (PBS). Both dynamic and equilibrium modulus of the samples immersed in Hexabrix? were significantly (p<0.05) lower than those of the samples immersed in PBS. Cartilage samples immersed in physiological saline solution showed load-induced cell death primarily in the superficial and middle zones. However, under high 8–10 MPa contact stresses, the samples immersed in full strength Hexabrix? showed significantly (p<0.05) higher number of dead cells than the samples compressed in physiological saline, especially in the deep zone of cartilage. In conclusion, excessive loading stresses followed by tissue creep might increase the risk for chondrocyte death in articular cartilage when immersed in hypertonic X-ray contrast agent, especially in the deep zone of cartilage.     

In vivo gene delivery of XIAP protects against myocardial apoptosis and infarction following ischemia/reperfusion in conscious rabbits

AimsWe tested the hypothesis that an in vivo gene delivery of the pro-survival protein XIAP (X-chromosome linked inhibitor of apoptosis protein) protects against myocardial apoptosis and infarction following ischemia/reperfusion.Main methodsNineteen rabbits were chronically instrumented with a hydraulic occluder placed around the circumflex coronary artery. Adenovirus harboring XIAP (Ad.XIAP; 1 × 10¹⁰ pfu/ml) or β-galactosidase (5 × 10⁹ pfu/ml), as a control, was constructed and transfected into the heart using a catheter placed into the left ventricle accompanied by cross-clamping. 1–2 weeks after gene delivery, myocardial ischemia was induced by a 30-min occlusion followed by reperfusion for four days. Protein expression was determined by Western blot and apoptosis (% of myocytes) was quantified by TUNEL staining.Key findingsMyocardial infarct size, expressed as a fraction of the area at risk, was reduced in Ad.XIAP (n = 5) compared to control (n = 7) rabbits (21 ± 3% vs. 30 ± 2%, p < 0.05). Apoptosis was reduced in Ad.XIAP rabbits compared to control rabbits (2.96 ± 0.68% vs. 8.98 ± 1.84%, p < 0.01). This was associated with an approximate 60% decrease in the cleaved caspase-3 level in Ad.XIAP rabbits compared to control rabbits.SignificanceThe current findings demonstrate that overexpression of XIAP via in vivo delivery in an adenovirus can reduce both myocardial apoptosis and infarction following ischemia/reperfusion, at least in part, due to the ability of XIAP to inhibit caspase-3. These findings confirm previous work suggesting a link between myocardial apoptosis and infarction i.e., anti-apoptotic therapy was effective in reducing myocardial infarct size.     

Nanomechanical phenotype of chondroadherin-null murine articular cartilage

Chondroadherin (CHAD), a class IV small leucine rich proteoglycan/protein (SLRP), was hypothesized to play important roles in regulating chondrocyte signaling and cartilage homeostasis. However, its roles in cartilage development and function are not well understood, and no major osteoarthritis-like phenotype was found in the murine model with CHAD genetically deleted (CHAD^−/−). In this study, we used atomic force microscopy (AFM)-based nanoindentation to quantify the effects of CHAD deletion on changes in the biomechanical function of murine cartilage. In comparison to wild-type (WT) mice, CHAD-deletion resulted in a significant ≈ 70–80% reduction in the indentation modulus, E_ind, of the superficial zone knee cartilage of 11 weeks, 4 months and 1 year old animals. This mechanical phenotype correlates well with observed increases in the heterogeneity collagen fibril diameters in the surface zone. The results suggest that CHAD mainly plays a major role in regulating the formation of the collagen fibrillar network during the early skeletal development. In contrast, CHAD-deletion had no appreciable effects on the indentation mechanics of middle/deep zone cartilage, likely due to the dominating role of aggrecan in the middle/deep zone. The presence of significant rate dependence of the indentation stiffness in both WT and CHAD^−/− knee cartilage suggested the importance of both fluid flow induced poroelasticity and intrinsic viscoelasticity in murine cartilage biomechanical properties. Furthermore, the marked differences in the nanomechanical behavior of WT versus CHAD^−/− cartilage contrasted sharply with the relative absence of overt differences in histological appearance. These observations highlight the sensitivity of nanomechanical tools in evaluating structural and mechanical phenotypes in transgenic mice.     

Conceptualisation of articular cartilage as a giant reverse micelle: A hypothetical mechanism for joint biocushioning and lubrication

Phospholipid (PL) molecules form the main structure of the membrane that prevents the direct contact of opposing articular cartilage layers. In this paper we conceptualise articular cartilage as a giant reverse micelle (GRM) in which the highly hydrated three-dimensional network of phospholipids is electrically charged and able to resist compressive forces during joint movement, and hence loading. Using this hypothetical base, we describe a hydrophilic–hydrophilic (HL–HL) biopair model of joint lubrication by contacting cartilages, whose mechanism is reliant on lamellar cushioning. To demonstrate the viability of our concept, the electrokinetic properties of the membranous layer on the articular surface were determined by measuring via microelectrophoresis, the adsorption of ions H, OH, Na and Cl on phospholipid membrane of liposomes, leading to the calculation of the effective surface charge density. The surface charge density was found to be −0.08 ± 0.002 cm⁻² (mean ± S.D.) for phospholipid membranes, in 0.155 M NaCl solution and physiological pH. This value was approximately five times less than that measured in 0.01 M NaCl. The addition of synovial fluid (SF) to the 0.155 M NaCl solution reduced the surface charge density by 30% which was attributed to the binding of synovial fluid macromolecules to the phospholipid membrane. Our experiments show that particles charge and interact strongly with the polar core of RM. We demonstrate that particles can have strong electrostatic interactions when ions and macromolecules are solubilized by reverse micelle (RM). Since ions are solubilized by reverse micelle, the surface entropy influences the change in the charge density of the phospholipid membrane on cartilage surfaces. Reverse micelles stabilize ions maintaining equilibrium, their surface charges contribute to the stability of particles, while providing additional screening for electrostatic processes.     

Lower limb joint work and joint work contribution during downhill and uphill walking at different inclinations

Work performance and individual joint contribution to total work are important information for creating training protocols, but were not assessed so far for sloped walking. Therefore, the purpose of this study was to analyze lower limb joint work and joint contribution of the hip, knee and ankle to total lower limb work during sloped walking in a healthy population. Eighteen male participants (27.0 ± 4.7 yrs, 1.80 ± 0.05 m, 74.5 ± 8.2 kg) walked on an instrumented ramp at inclination angles of 0°, ±6°, ±12° and ±18° at 1.1 m/s. Kinematic and kinetic data were captured using a motion-capture system (Vicon) and two force plates (AMTI). Joint power curves, joint work (positive, negative, absolute) and each joint’s contribution to total lower limb work were analyzed throughout the stance phase using an ANOVA with repeated measures. With increasing inclination positive joint work increased for the ankle and hip joint and in total during uphill walking. Negative joint work increased for each joint and in total work during downhill walking. Absolute work was increased during both uphill (all joints) and downhill (ankle & knee) walking. Knee joint contribution to total negative and absolute work increased during downhill walking while hip and ankle contributions decreased. This study identified, that, when switching from level to a 6° and from 6° to a 12° inclination the gain of individual joint work is more pronounced compared to switching from 12° to an 18° inclination. The results might be used for training recommendations and specific training intervention with respect to sloped walking.