The effects of lyophilization on flexural stiffness of extrasynovial and intrasynovial tendon

Tendon or ligament reconstructions often use autologous or allogenic tendons from either extrasynovial or intrasynovial sources. Allograft tendons must be lyophilized for preservation before transplantation, a process which can impact mechanical properties of the graft. Reconstituted graft properties that are similar to native tendon are desirable. Although tensile and compressive properties of tendons have been investigated, there is a paucity of information describing flexural properties of tendon, which can impact the gliding resistance. This study aims to design a testing method to quantify tendon flexural modulus, and investigate the effects of lyophilization/rehydration procedures on tendon flexibility. A total of 20 peroneus longus tendons (extrasynovial) and 20 flexor digitorum profundus tendons (intrasynovial) were collected. Ten of each tendon were processed with 5 freeze–thaw cycles followed by lyophilization and rehydration with saline solution (0.9%). Bend testing was conducted on tendons to quantify the flexural modulus with and without processing. As canine FDP tendons contain fibrous and fibrocartilaginous tissue regions, the flexural moduli were measured in both regions. Flexural modulus of rehydrated, lyophilized extrasynovial PL tendon was significantly lower than that of similarly processed intrasynovial FDP tendon (p < 0.001). Flexural moduli of both the fibrocartilaginous and non-fibrocartilaginous regions of intrasynovial tendon significantly increased after lyophilization (p < 0.001). The flexural modulus of the fibrocartilaginous region was significantly higher than that of the non-fibrocartilaginous region in intrasynovial tendon (p < 0.001). Lyophilization significantly increases the flexural modulus of extrasynovial and intrasynovial tendons, and flexural modulus differs significantly between these two tendon types. Increases in stiffness caused by lyophilization may impact the mechanical performance of the allograft in vivo.     

Kinetics of crimp and slope grip in rock climbing

The aim was to investigate differences of the kinetics of the crimp and the slope grip used in rock climbing. Nine cadaver fingers were prepared and fixated with the proximal phalanx in a frame. The superficial (FDS) and deep (FDP) flexor tendons were loaded selectively and together with 40 N in the crimp grip (PIP joint flexed 90°/DIP joint hyperextended) and the slope grip position (<25° flexed/50° flexed respectively). Five different grip sizes were tested and the flexion force which was generated to the grip was measured. In the crimp grip the FDP generated more flexion force in small sized holds whereas the FDS generated more force in the larger holds. During the slope grip the FDP was more effective than the FDS. While both tendons were loaded, the flexion force was always greater during crimp grip compared with the slope grip. The FDP seems to be most important for very small holds using the crimp grip but also during slope grip holds whereas the FDS is more important for larger flat holds.     

The Effect of Lubricin on the Gliding Resistance of Mouse Intrasynovial Tendon

The purpose of this study was to investigate the role of lubricin on the gliding resistance of intrasynovial tendons by comparing lubricin knockout, heterozygous, and wild type mice. A total of thirty-six deep digital flexor (DDF) tendons in the third digits of each hind paw from eighteen adult mice were used, including six lubricin knockout mice (Prg4 –/–), six heterozygous mice (Prg4 +/–), and six wild type mice (Prg4 +/+). The tendon gliding resistance was measured using a custom-made device. Tendon structural changes were evaluated by scanning electron and light microscopy. The gliding resistance of intrasynovial tendons from lubricin knockout mice was significantly higher than the gliding resistance of either wild type or heterozygous mice. The surface of the lubricin knockout tendons appeared to be rougher, compared to the wild type and heterozygous tendons. Synovial hyperplasia was found in the lubricin knockout mice. Cartilage-like tissue was found in the tendon and pulley of the lubricin knockout mice. Our findings confirm the importance of lubricin in intrasynovial tendon lubrication. This knockout model may be useful in determining the effect of lubricin on tendon healing and the response to injury.     

Compressive properties of cd-HA-gelatin modified intrasynovial tendon allograft in canine model in vivo

Although we sometimes use the intrasynovial tendon allograft as a donor, the gliding ability of allograft prepared by lyophilization is significantly decreased. The gliding ability of the grafted tendon after tendon reconstruction is very important because the high gliding resistance causes more adhesion and leads to poor clinical results. We recently revealed that tendon surface treatment with a carbodiimide derivatized HA (cd-HA)-gelatin mixture for intrasynovial tendon allograft significantly improved its gliding ability. The purpose of this study was to investigate whether this cd-HA-gelatin treatment affects the tendon mechanical property or not. A total of 40 flexor digitorum profundus (FDP) tendons from canines were evaluated for compressive property by using indentation test. Indentation stiffness was measured for normal tendon, rehydrated tendon after lyophilization, rehydrated tendon after lyophilization that was implanted 6 weeks in vivo, and cd-HA treated rehydrated tendon after lyophilization that was implanted 6 weeks in vivo. The results for all groups showed no significant difference in the tendon compressive properties. The findings of these results demonstrate that cd-HA treatment for intrasynovial tendon allograft is an excellent method to improve the tendon gliding ability after lyophilization without changing the compressive property of donor tendon.     

Effects of gamma irradiation and repetitive freeze-thaw cycles on the biomechanical properties of human flexor digitorum superficialis tendons

An increasing number of tissue banks have begun to focus on gamma irradiation and freeze-thaw in the reconstruction of anterior cruciate ligaments using allografts. The purpose of this study was to evaluate the biomechanical properties of human tendons after exposure to gamma radiation and repeated freeze-thaw cycles and to compare them with fresh specimens. Forty flexor digitorum superficialis tendons were surgically procured from five fresh cadavers and divided into four groups: fresh tendon, gamma irradiation, freeze-thaw and gamma irradiation+freeze-thaw. The dose of gamma irradiation was 25 kGy. Each freeze-thaw cycle consisted of freezing at -80 °C for 7 day and thawing at 25 °C for 6 h. These tendons underwent 4 freeze-thaw cycles. Biomechanical properties were analyzed during load-to-failure testing. The fresh tendons were found to be significantly different in ultimate load, stiffness and ultimate stress relative to the other three groups. The tendons of the gamma+freeze-thaw group showed a significant decrease in ultimate load, ultimate stress and stiffness compared with the other three groups. Gamma irradiation and repeated freezing-thawing (4 cycles) can change the biomechanical properties. However, no significant difference was found between these two processes on the effect of biomechanical properties. It is recommended that gamma irradiation (25 kGy) and repetitive freeze-thaw cycles (4 cycles) should not be adopted in the processing of the allograft tendons.     

Study on the physiology of diapause, cold hardiness and supercooling point of overwintering pupae of the pistachio fruit hull borer, Arimania comaroffi

The pistachio fruit hull borer, Arimania comaroffi (Ragonot) (Lepidoptera: Pyralidae), is a key pest of pistachio orchards in Iran. This pest passes the winter as diapausing pupae. In this study, some physiological changes in relation to environmental temperature were investigated in field collected pupae. The relationship between supercooling point, cold hardiness and physiological changes of a wild population of this pest was also investigated. The glycogen content decreased with decrease in environmental temperature. Decrease in glycogen content was proportional to increase in total body sugar, trehalose, myo-inositol and sorbitol contents. In January with mean ambient temperature of 5.4°C, glycogen (5 mg/g fresh body weight) content was at the lowest level whereas total body sugar (10.3 mg/g fresh body weight), trehalose (8.6 mg/g fresh body weight), myo-inositol (5.3 mg/g fresh body weight) and sorbitol (2.6 mg/g fresh body weight) were at the highest levels. Total body sugar, trehalose, myo-inositol and sorbitol contents increased as mean temperature decreased from 22.7°C in October to 5.4°C in January. Total body lipid decreased during overwintering and reached to the lowest level at the end of March. Supercooling points were decreased from October to January and reached to the lowest level (-16°C) in January with minimum ambient temperature of -10°C. Survival at low temperature after 24 h was also greatest in January with 72% survival at -10°C, 39% survival at -15°C and 0% survival at -20°C. Increase in temperature from February onward, was proportional with increase in supercooling points and decrease in survival rate. Regardless of sampling date, all pupae died after 24 h at -20°C, whereas none pupae died after 24 h at -5°C. This indicates that this insect is freeze-intolerant.     

Differential proteomic response of rice (Oryza sativa) leaves exposed to high- and low-temperature stress

Global mean surface temperature has been predicted to increase by 1.8-4°C within this century, accompanied by an increase in the magnitude and frequency of extreme temperature events. Developing rice cultivars better adapted to non-optimal temperatures is essential to increase rice yield in the future and, hence, understanding the molecular response of rice to temperature stress is necessary. In this study, we investigated the proteomic responses of leaves of 24-day-old rice seedlings to sudden temperature changes. Rice seedlings grown at 28/20°C (day/night) were subjected to 3-day exposure to 12/5°C or 20/12°C (day/night) for low-temperature stress, and 36/28°C or 44/36°C (day/night) for high-temperature stress, followed by quantitative label-free shotgun proteomic analysis on biological triplicates of each treatment. Out of over 1100 proteins identified in one or more temperature treatments, more than 400 were found to be responsive to temperature stress. Of these, 43, 126 and 47 proteins were exclusively found at 12/5, 20/12 and 44/36°C (day/night), respectively. Our results showed that a greater change occurs in the rice leaf proteome at 20/12°C (day/night) in comparison to other non-optimal temperature regimes. In addition, our study identified more than 20 novel stress-response proteins.     

Effects of temperature and dietary protein level on hepatic oxidative status of Senegalese sole juveniles (Solea senegalensis)

Effects of 55 and 45% dietary protein levels (55P and 45P diets, respectively) and temperature (12 and 18°C) on hepatic activity of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase (GR), glucose-6-phosphate dehydrogenase and lipid peroxidation (LPO) levels of Solea senegalensis juveniles were studied. Further, effects of acute thermal shocks provoked by a drop (18°C to 12°C) or a rise (12°C to 18°C) of water temperature on sole oxidative state was also evaluated. Dietary protein reduction increased LPO levels though no major alterations were found on antioxidant enzyme activities between dietary treatments. At 12°C GR activity was higher and SOD activity was lower than 18°C but LPO levels were not affected. In both thermal shock cases, LPO levels increased in 55P group, probably due to insufficient antioxidant enzyme activation. In contrast, fish of 45P group under acute exposition to warmer and colder temperature exhibited no substantial changes and a significant decrease on LPO levels, respectively, along with no major changes in antioxidant enzymes. Overall, results suggest that independently of rearing temperatures 45P group was more susceptible to oxidative stress than 55P group. Thermal shock either due to rise or drop of temperature seemed to induce oxidative stress in 55P group.     

Estimation of finger muscle tendon tensions and pulley forces during specific sport-climbing grip techniques

The present work displayed the first quantitative data of forces acting on tendons and pulleys during specific sport-climbing grip techniques. A three-dimensional static biomechanical model was used to estimate finger muscle tendon and pulley forces during the "slope" and the "crimp" grip. In the slope grip the finger joints are flexed, and in the crimp grip the distal interphalangeal (DIP) joint is hyperextended while the other joints are flexed. The tendons of the flexor digitorum profundus and superficialis (FDP and FDS), the extensor digitorum communis (EDC), the ulnar and radial interosseus (UI and RI), the lumbrical muscle (LU) and two annular pulleys (A2 and A4) were considered in the model. For the crimp grip in equilibrium conditions, a passive moment for the DIP joint was taken into account in the biomechanical model. This moment was quantified by relating the FDP intramuscular electromyogram (EMG) to the DIP joint external moment. Its intensity was estimated at a quarter of the external moment. The involvement of this parameter in the moment equilibrium equation for the DIP joint is thus essential. The FDP-to-FDS tendon-force ratio was 1.75:1 in the crimp grip and 0.88:1 in the slope grip. This result showed that the FDP was the prime finger flexor in the crimp grip, whereas the tendon tensions were equally distributed between the FDP and FDS tendons in the slope grip. The forces acting on the pulleys were 36 times lower for A2 in the slope grip than in the crimp grip, while the forces acting on A4 were 4 times lower. This current work provides both an experimental procedure and a biomechanical model that allows estimation of tendon tensions and pulley forces crucial for the knowledge about finger injuries in sport climbing.     

Effect of temperature level on thermal acclimation in Large White growing pigs

The effect of temperature level (24°C, 28°C, 32°C or 36°C) on performance and thermoregulatory response in growing pigs during acclimation to high ambient temperature was studied on a total of 96 Large White barrows. Pigs were exposed to 24°C for 10 days (days -10 to -1, P0) and thereafter to a constant temperature of 24°C, 28°C, 32°C or 36°C for 20 days. Pigs were housed in individual metal slatted pens, allowing a separate collection of faeces and urine and given ad libitum access to feed. Rectal (RT) and cutaneous (CT) temperatures and respiration rate (RR) were measured three times daily (0700, 1200 and 1800 h) every 2 to 3 days during the experiment. From day 1 to 20, the effect of temperature on average daily feed intake (ADFI) and BW gain (average daily gain, ADG) was curvilinear. The decrease of ADFI averaged 90 g/day per °C between 24°C and 32°C and 128 g/day per °C between 32°C and 36°C. The corresponding values for ADG were 50 and 72 g/day per °C, respectively. The 20 days exposure to the experimental temperature was divided in two sub-periods (P1 and P2, from day 1 to 10 and from day 11 to 20, respectively). ADFI was not affected by duration of high-temperature exposure (i.e. P2 v. P1). The ADG was not influenced by the duration of exposure at 24°C and 28°C groups. However, ADG was higher at P2 than at P1 and this effect was temperature dependent (+130 and +458 g/day at 32°C and 36°C, respectively). In P2 at 36°C, dry matter digestibility significantly increased (+2.1%, P < 0.01); however, there was no effect of either duration or temperature on the digestibility of dry matter at group 24°C and 32°C. RT, CT and RR were measured three times daily (0700, 1200 and 1800 h) every 2 to 3 days during the experiment. Between 28°C and 36°C, RT and CT were lower during P2 than during P1 (-0.20°C and -0.23°C; P < 0.05), whereas RR response was not affected by the duration of exposure whatever the temperature level. In conclusion, this study suggests that the effect of elevated temperatures on performance and thermoregulatory responses is dependent on the magnitude and the duration of heat stress.     

Ecologically relevant measures of the physiological tolerance of light brown apple moth, Epiphyas postvittana, to high temperature extremes

Invasive ectothermic species are limited in their geographic range expansion primarily by their capacity to withstand temperature extremes. Epiphyas postvittana is a highly polyphagous invasive leafroller that was discovered in California in 2006. To predict its potential range and future response to climate change, high temperature tolerance of this species was determined for all life stages and larval instars. Using the static method to estimate high temperature tolerance with response to probing as an endpoint, the mean time leading to 50% mortality (LT(50)) ranged from 45 to 187h at 32.3°C, 34 to 68h at 36°C, 11 to 21h at 38°C, and 1.2 to 5.6h at 40.4°C. There was no clear pattern in the relative tolerance of the life stages across the range of temperatures tested. For pupae and adults, gender did not influence the LT(50) values at any of the temperatures tested. For the larval instars, LT(50) values increased with increasing larval instar at the highest three temperatures while this trend was reversed for the lowest temperature (32.3°C). An analysis of LT(50) values obtained from acute responses to probing compared to subsequent survival to adult emergence, showed that chronic mortality severely affected all larval instars at three out of the four constant temperatures and resulted in 64-85% reduction in LT(50) values. No difference in acute and chronic mortality was found for exposure of the egg stage to high temperatures. These findings have important implications for predicting thermal limits and range expansions of insect species, since upper thermal tolerance could readily be overestimated from the use of ad hoc rather than ecologically relevant endpoint measurements such as survival to adult emergence.     

Effects of microhabitat,light and temperature on seed germination of a critically endangered Himalayan medicinal herb,Swertia chirayita: Conservation implications

Abstract

Swertia chirayita, a critically endangered medicinal herb, is being over-harvested in the wild. Understanding seed germination is a pre-requisite to ensure species conservation. The germination of seeds collected from six microhabitats was studied at 20°C, 25°C, and 30°C, both under a 14/10 h light/dark photoperiod and in continuous darkness. Two-way ANOVA indicated that microhabitat and temperature significantly affect seed germination, germination rate, germination recovery (GR), and GR rate. Overall, the seeds collected from under canopy showed a significantly (p < 0.05) higher germination than those from open habitats, at 20°C, 25°C, and 30°C (14/10 h light/dark photoperiod). Germination was negligible in continuous darkness but after transfer to a 14/10 h light/dark photoperiod, the seeds from under canopy significantly recovered at 20°C and at 25°C (p < 0.05), and showed the highest germination percentage compared to seeds collected from tree base, stump base, shrubberies, and grassy slope. Similarly, at 30°C, seeds from under canopy recorded the highest GR percentage. In general, seed germination, mean germination rate, seed GR, and GR rate were significantly greater (p < 0.05) at 25°C. Among the microhabitats tested, variation in GR rate was significant (p < 0.05). Seeds were confirmed to be positively photoblastic.     

Temperature induces trade-offs between development and starvation resistance in Aedes aegypti (L.) larvae

Heightened temperature increases the development rate of mosquitoes. However, in Aedes aegypti (Diptera: Culicidae), the larvae of which commonly experience limited access to food in urban habitats, temperature effects on adult production may also be influenced by changes in the capacity of larvae to survive without food. We carried out experiments to investigate the effects of temperatures increasing at intervals of 2 °C from 20 °C to 30 °C on the growth, maturation rate and longevity of optimally fed larvae placed in starvation. Overall, both growth rate and starvation resistance were lower in the first three larval instars (L1-L3) compared with L4, in which growth of >75% occurred. Although increasing the temperature reduced the duration of each instar, it had a U-shaped impact in terms of the effect of initial growth on starvation resistance, which increased from L1 to L2 at 20 °C and 30 °C, remained constant at 22 °C and 28 °C, and decreased at 24 °C and 26 °C. Growth from L2 to L3 significantly increased starvation resistance only from 26 °C to 30 °C. Increased temperature (>22 °C) consistently reduced starvation resistance in L1. In L2-L4, increments of 2 °C decreased starvation resistance between 20 °C and 24 °C, but had weaker and instar-specific effects at >24 °C. These data show that starvation resistance in Ae. aegypti depends on both instar and temperature, indicating a trade-off between increased development rate and reduced starvation survival of early-instar larvae, particularly in the lower and middle temperatures of the dengue-endemic range of 20-30 °C. We suggest that anabolic and catabolic processes in larvae have distinct temperature dependencies, which may ultimately cause temperature to modify the density regulation of Ae. aegypti populations.     

Temperature dependence of force, velocity, and processivity of single kinesin molecules

Using the bead assay in optical microscopy equipped with optical tweezers, we have examined the effect of temperature on the gliding velocity, force, and processivity of single kinesin molecules interacting with a microtubule between 15 and 35 degrees C. The gliding velocity increased with the Arrhenius activation energy of 50 kJ/mol, consistent with the temperature dependence of the microtubule-dependent ATPase activity. Also, the average run length, i.e., a measure of processivity of kinesin, increased on increasing temperature. On the other hand, the generated force was independent of temperature, 7.34 +/- 0.33 pN (average +/- S.D., n = 70). The gliding velocities decreased almost linearly with an increase in force irrespective of temperature, implying that the efficiency of mechano-chemical energy conversion is maintained constant in this temperature range. Thus, we suggest that the force generation is attributable to the temperature-insensitive nucleotide-binding state(s) and/or conformational change(s) of kinesin-microtubule complex, whereas the gliding velocity is determined by the ATPase rate.     

Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters

So far, there has been a discrepancy between the velocities of kinesin-dependent microtubule motility measured in vitro and within cells. By changing ATP, Mg(2+), and kinesin concentrations, pH and ionic strength, we tried to find conditions that favour microtubule gliding across kinesin-covered glass surfaces. For porcine brain kinesin, we found that raising the molar Mg(2+)/ATP ratio can substantially elevate gliding velocity. Gliding became also faster after temperature elevation or lowering the number of kinesin molecules bound to the glass surface. The highest mean gliding velocity (1.8 microm/s+/-0.09 microm/s), approaching velocities measured for anterograde transport in vivo, was achieved by combination of favourable factors (2.5 m m ATP, 12.5 m m Mg(2+), 37 degrees C, 450 kinesin molecules/microm(2)).     

Relative humidity preference and survival of starved Formosan subterranean termites (Isoptera: Rhinotermitidae) at various temperature and relative humidity conditions

Foraging groups of Formosan subterranean termites, Coptotermes formosanus Shiraki were tested for their relative humidity (RH) preference in a humidity gradient arena in the laboratory at a constant temperature of 26°C. Five RH levels (9%, 33%, 53%, 75%, and 98%) were maintained in the test arena comprising of a series of closed containers by using dry silica gel, saturated salt solutions, or distilled water alone. Termites gradually aggregated to the highest RH chamber in the arena. After 1 h, a significantly greater percentage of termites (≈46%) aggregated to the highest RH chamber (98%) than to the lower RH chambers (≤75%). After 12 h, > 97% of the termites aggregated to the 98% RH chamber. In survival tests, where termites were exposed to 15 combinatorial treatments of five RH levels (9%, 33%, 53%, 75%, and 98%) and three temperatures (20°C, 28°C, and 36°C) for a week, the survival was significantly influenced by RH, temperature, and their interaction. A significantly higher mortality was observed on termites exposed to ≤75% RH chambers than to 98% RH chamber at the three temperatures and significantly lower survival was found at 36°C than at 28°C or 20°C. The combination of temperature and RH plays an important role in the survival of C. formosanus.     

Resistance forces acting on suture needles

Understanding the resistance forces encountered by a suture needle during tissue penetration is important for the development of robotic surgical devices and virtual reality surgical simulators. Tensile forces applied to skin and tendon during suturing were measured. Fresh sheep achilles tendons were tensioned with a static load 4.9 N, 9.8 N or 19.6 N and sheepskin with 0.98 N, 2.9 N or 4.9 N static load. A straight 2/0 cutting suture needle in series with a load cell on a materials testing machine penetrated the tissue at 90 degrees with a velocity of 1, 5 or 10mm/s for each tissue tension (n=5). Continuous load versus displacement data was obtained and penetration load and stiffness were noted. The load versus displacement curve for skin during needle penetration demonstrated two characteristic peaks, corresponding to initial penetration and emergence of needle from the undersurface of the tissue. Increasing the tension within the tissue (skin and tendon) increased the amount of force required to penetrate the tissue with a suture needle (p<0.05). Needle displacement rate did not affect the resistance to needle penetration (p<0.05). This study provides a simple model for measuring force-feedback during needle penetration of soft tissues and is a good starting point for future studies of the penetration resistance properties of human tissues.     

Nuclear translocation of lymphoblast glucocorticoid receptors in childhood leukemia does not predict steroid responsiveness

Nuclear translocation of glucocorticoid receptors (GR) was measured in lymphoblasts from 45 children with acute lymphoblastic leukemia (ALL). The percent of total GR translocated to the nucleus ranged from 0 to 100% (mean, 59%) and was independent of the total GR level. Neither the extent of GR nuclear translocation nor the absolute amount of GR translocated at 4 or 25 degrees C correlated with glucocorticoid response in 13 patients with single-agent glucocorticoid trial. These results indicate that defects in nuclear translocation of GR are not a common cause for steroid resistance in childhood ALL.     

Kinesin velocity increases with the number of motors pulling against viscoelastic drag

Although the properties of single kinesin molecular motors are well understood, it is not clear whether multiple motors pulling a single vesicle in a cell cooperate or interfere with one another. To learn how small numbers of motors interact, microtubule gliding assays were carried out with full-length Drosophila kinesin in a novel motility medium containing xanthan, a stiff, water-soluble polysaccharide. At 2 mg/ml xanthan, the zero-shear viscosity of this medium is 1,000 times the viscosity of water, similar to cellular viscosity. To mimic the rheological drag force on the motors when attached to a vesicle in a cell, we attached a 2 μm bead to one end of the microtubule (MT). During gliding assays in our novel medium, the moving bead exerted a drag force of 4–15 pN on the kinesins pulling the MT. The velocity of MTs with an attached bead increased with MT length and with kinesin concentration. The increase with MT length arose because the number of motors is directly proportional to MT length. Our results show that small numbers of kinesins cooperate constructively when pulling against a viscoelastic drag. In the absence of a bead but still in the viscous medium, MT velocity was independent of MT length and kinesin concentration because the thin MT, like a snake moving through grass, was able to move between xanthan molecules with little resistance. A minimal shared-load model in which the number of motors is proportional to MT length fits the observed dependence of gliding velocity on MT length and kinesin concentration.