The effect of different methods of cross-linking of collagen on its dielectric properties

This paper reports on the effect of different methods of collagen cross-linking on its dielectric properties. In order to obtain collagen-hyaluronic acid (HA) scaffolds, collagen was first dehydrated by a combination of thermal and vacuum drying (DHT) and then treated with the chemical reagent carbodiimide (EDC/NHS) for final cross-linking. The measurements of the relative permittivity varepsilon' and the dielectric loss varepsilon' for all materials were carried over the frequency range of 10 Hz-100 kHz and at temperatures from 22 to 260 degrees C. The results for these samples reveal distinct relaxation processes at low temperatures, below 140 degrees C and at higher temperatures as broad peak around 230 degrees C. The first and second relaxation are associated with changes in the secondary structure of collagen accompanied by the release of water and with the denaturation of dry collagen, respectively. The influence of cross-linking on the permittivity of collagen is significant over the entire temperature range.     

Fracture toughness and work of fracture of hydrated, dehydrated, and ashed bovine bone

Bone, a tri-phase composite, consists of nano-sized apatite minerals, an organic component, and water. Heat-treated bovine cortical bone has been proposed as a candidate for void-filling bone substitute. However, the toughness of heat-treated bone is not yet fully studied. Fracture toughness (K(c)) and work of fracture (W(f)) of hydrated, dehydrated, and ashed bovine bone were estimated using a single-edge V-notched beam method. Thermal gravimetric analysis and differential thermal analysis were used to determine the temperature at which the organics and water were removed. Dehydrated specimens were obtained by placing the samples in a 60 degrees C vacuum oven for 24h or a 110 degrees C furnace for 2h. Ashed specimens were obtained by heat-treating samples at 600 degrees C for 24h. K(c) of bovine specimens decreased from 5.5MPa.m(1/2) for hydrated bone, to 3.8MPa.m(1/2) for dehydrated specimens, and to 0.36MPa.m(1/2) for ashed specimens. W(f) decreased from 7.1 to 1.1kJ/m(2) for dehydrated specimens, and to 0.04kJ/m(2) for ashed specimens. The main reasons for the significant decreases in K(c) and W(f) may be attributed to water's ability in stabilizing collagen structure and to the organics' ability in making bone more ductile. Because of the large decrease in fracture toughness and work of fracture, we suggest that ashed bone is not appropriate for load-bearing bone substitute in areas where bone experiences loadings in flexure.     

Bone material properties and mineral matrix contributions to fracture risk or age in women and men

The strength of bone is related to its mass and geometry, but also to the physical properties of the tissue itself. Bone tissue is composed primarily of collagen and mineral, each of which changes with age, and each of which can be affected by pharmaceutical treatments designed to prevent or reverse the loss of bone. With age, there is a decrease in collagen content, which is associated with an increased mean tissue mineralization, but there is no difference in cross-link levels compared to younger adult bone. In osteoporosis, however, there is a decrease in the reducible collagen cross-links without an alteration in collagen concentration; this would tend to increase bone fragility. In older people, the mean tissue age (MTA) increases, causing the tissue to become more highly mineralized. The increased bone turnover following menopause may reduce global MTA, and would reduce overall tissue mineralization. Bone strength and toughness are positively correlated to bone mineral content, but when bone tissue becomes too highly mineralized, it tends to become brittle. This reduces its toughness, and makes it more prone to fracture from repeated loads and accumulated microcracking. Most approved pharmaceutical treatments for osteoporosis suppress bone turnover, increasing MTA and mineralization of the tissue. This might have either or both of two effects. It could increase bone volume from refilling of the remodeling space, reducing the risk for fracture. Alternatively, the increased MTA could increase the propensity to develop microcracks, and reduce the toughness of bone, making it more likely to fracture. There may also be changes in the morphology of the mineral crystals that could affect the homogeneity of the tissue and impact mechanical properties. These changes might have large positive or negative effects on fracture incidence, and could contribute to the paradox that both large and small increases in density have about the same effect on fracture risk. Bone mineral density measured by DXA does not discriminate between density differences caused by volume changes, and those caused by changes in mineralization. As such, it does not entirely reflect material property changes in aging or osteoporotic bone that contribute to bone's risk for fracture.     

Protein-reactive, thermoresponsive copolymers with high flexibility and biodegradability

A family of injectable, biodegradable, and thermosensitive copolymers based on N-isopropylacrylamide, acrylic acid, N-acryloxysuccinimide, and a macromer polylactide-hydroxyethyl methacrylate were synthesized by free radical polymerization. Copolymers were injectable at or below room temperature and formed robust hydrogels at 37 degrees C. The effects of monomer ratio, polylactide length, and AAc content on the chemical and physical properties of the hydrogel were investigated. Copolymers exhibited lower critical solution temperatures (LCSTs) from 18 to 26 degrees C. After complete hydrolysis, hydrogels were soluble in phosphate buffered saline at 37 degrees C with LCSTs above 40.8 degrees C. Incorporation of type I collagen at varying mass fractions by covalent reaction with the copolymer backbone slightly increased LCSTs. Water content was 32-80% without collagen and increased to 230% with collagen at 37 degrees C. Hydrogels were highly flexible and relatively strong at 37 degrees C, with tensile strengths from 0.3 to 1.1 MPa and elongations at break from 344 to 1841% depending on NIPAAm/HEMAPLA ratio, AAc content, and polylactide length. Increasing the collagen content decreased both elongation at break and tensile strength. Hydrogel weight loss at 37 degrees C was 85-96% over 21 days and varied with polylactide content. Hydrogel weight loss at 37 degrees C was 85-96% over 21 days and varied with polylactide content. Degradation products were shown to be noncytotoxic. Cell adhesion on the hydrogels was 30% of that for tissue culture polystyrene but increased to statistically approximate this control surface after collagen incorporation. These newly described thermoresponsive copolymers demonstrated attractive properties to serve as cell or pharmaceutical delivery vehicles for a variety of tissue engineering applications.     

Responses to desiccation in four Coleopterans from sub-Antarctic South Georgia

RATES OF WATER LOSS WERE DETERMINED FOR FOUR COLEOPTERA SPECIES: the herbivores Hydromedion sparsutum, Perimylops antarcticus (Family Perimylopidae) and the carnivores Trechisibus antarcticus, Oopterus soledadinus (Family Carabidae) collected during summer from a range of terrestrial habitats at South Georgia. A recording microbalance enabled measurement of individual weight loss with time in <5% r.h. at 10, 20, 30 and 35 degrees C. Adults of T. antarcticus had significantly higher rates of water loss than any other species over all temperatures. Individuals of both herbivores exhibited the slowest water loss rates under the experimental conditions. Within species, rates at 10 or 20 degrees C were slower than at the higher temperatures. Adult P. antarcticus had significantly greater amounts of body water than adult H. sparsutum for each of the four temperatures. Within species and life-stages of both herbivores, body water contents after drying at 10 degrees C were significantly lower than individuals dried at 30-35 degrees C, but no such differences were observed for the carnivores. At each temperature, rates of water loss were negatively correlated with initial live weight in all four species, but this was not the case within species or between adults and larvae. Maximum survival times during desiccation declined as temperature increased, but did not differ between species at 10 degrees C. Over 30-35 degrees C, survival times of both herbivores were significantly longer than either of the carnivores. Smaller insects (e.g. the carabids) had faster rates of water loss than the larger perimylopids under the same environmental conditions. The latter had greater resistance to desiccation than the former. It is suggested that the larger body water content of P. antarcticus enables it to resist desiccation more than the other three species, which correlates with its ecological distribution. Differences in water contents after drying individuals at low and high temperatures may be caused either by the water binding properties of cells and tissues or by reduction in energy stores in order to maintain metabolism at lower environmental temperatures causing a body weight loss. Whilst both herbivores show some physiological adaptations to drying conditions, it is suggested that the two carnivorous beetles may have adapted behaviourally to the South Georgia environment.     

Effect of temperature on the fracture toughness of compact bone

Bone is a composite composed mainly of organics, minerals, and water. Many researchers have studied effects such as crack velocity, density, orientation, storage media, porosity, and age on the fracture toughness (K(C), also called critical stress intensity factor) of compact bone. Most of these studies were conducted at room temperature. Considering that the body temperature of animals is greater than room temperature, and that bone has a large volumetric percentage of organics and water (generally, 55-65%), it is hypothesized that temperature has a significant effect on the fracture toughness of compact bone. Single-edge V-notched (SEVN) specimens were prepared to measure the fracture toughness of bovine femur and manatee rib in water at 0, 10, 23, 37, and 50 degrees C in four-point flexure. The fracture toughness values of bovine femur and manatee rib were found to decrease from 7.0 to 4.3MPam(1/2) and from 5.5 to 4.0MPam(1/2), respectively, as temperature increased over a temperature range of 50 degrees C. The results support the hypothesis that temperature has a significant effect on the fracture toughness of compact bone. Therefore, we suggest that study on fracture toughness of bone should be done at physiologically relevant temperatures.     

Effect of drying on the biological activities of a red microalgal polysaccharide

The red microalga Porphyridium sp. produces a polysaccharide exhibiting a variety of biological activities with potential for medical and cosmetic uses. For this reason, it is important that the drying process, which is the end point of production, should not destroy the natural characteristics of the material. The objective of this study was to evaluate the effect of drying at temperatures ranging from 40 to 140 degrees C on the bioactivities of the polysaccharide. Drying the polysaccharide at temperatures above 90 degrees C caused a significant decline in its biological activities (antiviral and anti-cell proliferation) and reduced elasticity, viscosity, and intrinsic viscosity relative to lyophilized polysaccharide and to the starting product. The relationship between molecular weight and intrinsic viscosity indicated that the polysaccharide takes a rigid coil conformation, which stiffens as a result of drying. FTIR analysis revealed that drying caused both significant conformational alterations in the polymer chains and changes in the interaction between the polysaccharide and the glycoprotein to which it is noncovalently associated. Differential scanning calorimetry analysis of the water adsorbed on the charged groups of the polysaccharide showed that drying at higher temperatures increased the bound water content due to dissociation of the polymer chains. Thus, it is recommended that the polysaccharide be dried in a two-step process in which free water is removed by convection and bound freezing water is removed by lyphophilization.     

Cyclic gas exchange in the giant burrowing cockroach, Macropanesthia rhinoceros: effect of oxygen tension and temperature

The giant burrowing cockroach, Macropanesthia rhinoceros, is endemic to north-eastern Australia and excavates a permanent burrow up to 1m deep into soil. Using flow-through respirometry, we investigated gas exchange and water loss at three different oxygen tensions (21%, 10% and 2% at 20 degrees C) and temperatures (10, 20 and 30 degrees C at 21% oxygen). M. rhinoceros employ cyclic gas exchange (CGE) making the species by far the largest insect known to engage in discontinuous ventilation. CGE featured rhythmic bursts of CO(2) dispersed among inter-burst periods of reduced output. CGE was most commonly observed at 20 degrees C and degraded at <10% oxygen. Mild hypoxia (10% oxygen) resulted in a lengthening of the burst period by approximately two-fold; this result is complementary to oxygen consumption data that suggests that the burst period is important in oxygen uptake. When exposed to severe hypoxia (2% oxygen), CGE was degraded to a more erratic continuous pattern. Also, during severe hypoxia, total water loss increased significantly, although CO(2) release was maintained at the same level as in 21% oxygen. During CGE, an increase in temperature from 10 to 20 degrees C caused both water loss and CO(2) output to double; from 20 to 30 degrees C, CO(2) output again doubled but water loss increased by only 31%.     

Changes in body water and plasma constituents during bullfrog development: effects of temperature and hormones

The osmoregulatory responses to warmer temperatures and hormone treatment in cold-adapted (5 degrees C) Rana catesbeiana tadpoles and newly metamorphosed frogs were examined. Tadpoles transferred to 11 degrees C and 18 degrees C and left for 5 days lost 7% and 10% of their body weight. Plasma [Na+] was elevated 28% and 21%, respectively. Control (5 degrees C) animals maintained their body weight and plasma [Na+] constant. Daily treatment with either ovine prolactin (oPRL) or ovine growth hormone (oGH) prevented the weight loss and the increase in extracellular [Na+] that occurred when tadpoles were transferred to 18 degrees C. Neither propylthiouracil (PTU) nor arginine vasotocin (AVT) were effective in countering temperature-induced weight loss in tadpoles. Newly metamorphosed frogs transferred to 18 degrees C also lost weight; this was not prevented by daily treatment with saline, oPRL, oGH or PTU. However, in frogs treated daily with AVT, initial BW was regained by day 6. When warm-adapted (18 degrees C) tadpoles were treated daily for 18 days with saline, bPRL, bGH, thyroxine (T4), ergocornine, cortisol, or cortisol + T4, bPRL was most effective in retarding weight loss and maintaining body water content, whereas T4 + cortisol caused the greatest loss of weight and body water. By day 20, the correlations between weight loss and both body water content and hematocrit were highly significant. These data suggest that reported increases in plasma solute concentrations in larval amphibians may actually reflect decreases in extracellular fluid volume, rather than increased amounts of solutes, per se.     

Loss of selenium in drying and storage of agronomic plant species

Summary In two experiments with Se⁷⁵, loss of selenium from agricultural species was noted during both drying and storage. The loss of selenium during drying was to some extent overshadowed by the influence of self-absorption caused by the water in the fresh material. The results showed that even plant material of non-indicator plantslose volatile selenium at drying temperatures of 60°C or higher, and in some cases even at temperatures below 60°C. The results also showed that storage as briquettes gives the lowest storage loss of selenium.     

Dielectric characterization of neutralized and nonneutralized chitosan upon drying

Isothermal dielectric loss spectra of neutralized and nonneutralized chitosan were acquired in successive runs from -130 degrees C up to increasing final temperatures, in a frequency range between 20 Hz and 1 MHz. Essentially, three relaxation processes were detected in the temperature range covered: (i) a beta-wet process, detected when the sample has a higher water content that vanishes after heating to 150 degrees C; (ii) a beta process, which is located at temperatures below 0 degrees C, becoming better defined and maintaining its location after annealing at 150 degrees C independently of the protonation state of the amino side group; and (iii) a sigma process that deviates to higher temperatures with drying, being more mobile in the nonneutralized form. Moreover, in dried neutralized chitosan, a fourth process was detected in the low frequency side of the secondary beta process that diminishes after annealing. Whether this process is a distinct relaxation of the dried polymer or a deviated beta-wet process due to the loss of water residues achieved by annealing is not straightforward. Only beta and sigma processes persist after annealing at 150 degrees C. The changes in molecular mobility upon drying of these two relaxation processes were evaluated.     

Fermentation efficiency of thermally dried kefir

Three thermal drying methods (conventional, vacuum and convective) were used for drying of kefir biomass and their effect on cell viability, fermentation rate and other kinetic parameters of lactose and whey fermentation were studied. Convective drying rate was higher than conventional and even higher than vacuum at each studied temperature (28, 33 and 38 degrees C). After that, fermentations were performed by kefir biomass dried by the three drying methods. Ethanol concentration, ethanol productivity and ethanol yield are higher in whey fermentations performed by kefir biomass dried with convective drying method. Regarding lactic acid production, fermentation performed by kefir biomass dried with conventional drying method gave higher concentrations, compared to other drying methods. Storage of kefir biomass convectively dried at 33 degrees C for 4months, without any precaution decreases its fermentability and thus reduces ethanol (31%) and lactic acid productivity (20%), but remains a promising technology, since a significant part of its initial fermentative activity is retained.     

Thermal denaturation of UV-irradiated wet rat tail tendon collagen

The thermal helix-coil transition of UV irradiated collagen in rat tail tendon has been investigated by differential scanning calorimetry. During UVB irradiation the tendons were immersed in water to keep the collagen fibers in a fully hydrated condition at all times. UV irradiation induced changes in collagen which caused both stabilization and destabilization of the triple helix in fibers. The helix-coil transition for non-irradiated collagen occurred near 64 degrees C, for irradiated 1 and 3 h at 66 and 67 degrees C, respectively. After irradiating for longer times (20-66 h) the helix-coil transition peak occurred at much lower temperatures. The peak was very broad and suggested that collagen was reduced by UV to different polypeptides of different molecular weight and different lower thermal stabilities. It was caused by the disruption of a network of hydrogen-bonded water molecules surrounding the collagen macromolecule.     

Failure of mineralized collagen fibrils: modeling the role of collagen cross-linking

Experimental evidence demonstrates that collagen cross-linking in bone tissue significantly influences its deformation and failure behavior yet difficulties exist in determining the independent biomechanical effects of collagen cross-linking using in vitro and in vivo experiments. The aim of this study is to use a nano-scale composite material model of mineral and collagen to determine the independent roles of enzymatic and non-enzymatic cross-linking on the mechanical behavior of a mineralized collagen fibril. Stress–strain curves were obtained under tensile loading conditions without any collagen cross-links, with only enzymatic cross-links (modeled by cross-linking the end terminal position of each collagen domain), or with only non-enzymatic cross-links (modeled by random placement of cross-links within the collagen–collagen interfaces). Our results show enzymatic collagen cross-links have minimal effect on the predicted stress–strain curve and produce a ductile material that fails through debonding of the mineral–collagen interface. Conversely, non-enzymatic cross-links significantly alter the predicted stress–strain response by inhibiting collagen sliding. This inhibition leads to greater load transfer to the mineral, which minimally affects the predicted stress, increases modulus and decreases post-yield strain and toughness. As a consequence the toughness of bone that has more non-enzymatically mediated collagen cross-links will be drastically reduced.     

Salicylate hypothermia in rats exposed to hyperbaric air and helium.

Rectal temperatures of salicylate-treated and untreated rats were observed in 21-23 degrees C air at 1, 3, 6, and 8 ATA, in 21-23 degrees C helium at 1 and 6 ATA, and in 1 ATA thermal neutral air (28 degrees C). Significant dose-related temperature decreases occurred in 21-23 degrees C 1 ATA air with 180 and 300 mg/kg of salicylate; 60 mg/kg had no effect. However, in thermal neutral air, 300 mg/kg significantly elevated temperatures. Hyperbaric air and helium had temperature-lowering effects which were correlated with thermal properties of these environments, and in them the hypothermic effects of salicylate were similar to those in 1 ATA air, the total temperature decreases being the sum of the salicylate hypothermia and that caused by the hyperbaric air or helium. These additive temperature effects are unlike previously reported results in which the temperature lowering effects of 5 degrees C cold exposure and salicylate together were greater than the sum of the two individual effects.     

The structure of water absorbed in collagen. I. The dielectric properties

The dielectric constant and the loss factor of water in collagen are measured for various water, NaCl, and HCl contents at frequencies ranging from 10² to 10⁵ Hz and at temperatures ranging from ?70° to +23°C. For all measurements, both the dielectric constant and the loss decrease monotonically as the frequency increases, or the temperature decreases; the absence of a maximum in the loss curves as a function of temperature and frequency indicates an extremely broad spectrum of relaxation times. By shifting the curves obtained for the dielectric constant and the loss factor along the log–frequency axis, all data, obtained at different temperatures, can be represented on master curves valid for 23°C. In order to explain these results, water molecules are assumed to be hydrogen bonded to each other in long chains. All water molecules in a chain can, cooperatively, be oriented in two different directions along the channel, resulting in large, reversible, dipole moments. These chains are not rigid, but are flexible liquid-like structures. Diffusion of chains as entities is assumed to be the rate-limiting step for dipole reorientation. Although the rate of diffusion decreases inversely proportional to chain length, the activation energy is independent of chain length. At lower temperatures, diffusion becomes slower, until at the glass point, approximately ?100°C, it ceases.     

In vivo anti-osteoporotic activity of isotaxiresinol, a lignan from wood of Taxus yunnanensis

Isotaxiresinol, the main lignan isolated from the water extract of wood of Taxus yunnanensis, was investigated for its effect on bone loss, on serum biochemical markers for bone remodeling and on uterine tissue, using ovariectomized (OVX) rats as the model of postmenopausal osteoporosis. After oral administration of isotaxiresinol (50 and 100mg/kg/d) for 6 weeks, bone mineral content (BMC) and bone mineral density (BMD) in total and cortical bones were increased as compared to those of OVX control rats, and decreases of three bone strength indexes induced by OVX surgery were prevented. Serum biochemical markers for bone remodeling revealed that isotaxiresinol slightly increased bone formation and significantly inhibited bone resorption without side effect on uterine tissue. These results suggest that isotaxiresinol may be useful for treatment of postmenopausal osteoporosis, especially for prevention of bone fracture induced by estrogen deficiency.     

Effect of chemical modifications on the susceptibility of collagen to proteolysis. II. Dehydrothermal crosslinking.

Collagen was dehydrothermally treated (heat cured) by heating dry under vacuum at 60, 80, 100 and 120 degrees C. The change in stability was determined by subjecting to measurement of gross crosslinking, content of lysino-alanine and naturally occurring collagen crosslinks, shrinkage temperature (TM), susceptibility to digestion by lysosomal thiol proteases, and susceptibility to pepsin and trypsin. Morphological changes were examined by electron microscopy. The in vivo biodegradation of dehydrothermally treated collagen sponges was investigated using a rat lumbar muscle implantation model for up to 28 days. For all heat-cured collagens, the data strongly indicated that both crosslinking and denaturation/degradation was present in increasing quantities with increasing temperature of treatment, its level was too low (maximum 179 pmol mg-1) to account for the decreased solubility and increased molecular weight gross changes observed. Increasing resistance of treated collagen to both lysosomal cathepsins and pepsin correlated well with increased crosslinking and increasing temperature of the heat-curing process. However, increased denaturation/degradation of the collagen at higher temperatures was revealed by electrophoretic analysis, trypsin hydrolysis data and by electron microscopy. Differential scanning calorimetry (d.s.c.) correlated well with these results showing an increased level of denaturation in heated samples. The in vivo study showed little difference between control and heat-cured samples except for the material treated at 120 degrees C which was biodegraded in vivo at a significantly faster rate. The data shows, therefore, that crosslinking induced by the dehydrothermal treatment of collagen decreases its rate of proteolysis at acid pH in vitro. However, the simultaneous denaturation/degradation of the protein during the heat-cure process appears to be a more important factor in determining the fate of the material implanted into rat muscle.     

Time Related Changes of Mineral and Collagen and Their Roles in Cortical Bone Mechanics of Ovariectomized Rabbits

As cortical bone has a hierarchical structure, the macroscopic bone strength may be affected by the alterations of mineral crystal and collagen, which are main components of cortical bone. Limited studies focused on the time related alterations of these two components in osteoporosis, and their contributions to bone mechanics at tissue level and whole-bone level. Therefore, the purpose of this study was to elucidate the time related changes of mineral and collagen in cortical bone of ovariectomized (OVX) rabbits, and to relate these changes to cortical bone nanomechanics and macromechanics. 40 Rabbits (7-month-old) were randomly allocated into two groups (OVX and sham). OVX group received bilateral ovariectomy operation. Sham group received sham-OVX operation. Cortical bone quality of five rabbits in each group were assessed by DXA, μCT, nanoindentation, Fourier transform infrared (FTIR) spectroscopy and biomechanical tests (3-point bending of femoral midshaft) at pre-OVX, 4, 6, and 8 weeks after OVX. As time increased from pre-OVX to 8 weeks, the mineral to matrix ratio decreased with time, while both collagen crosslink ratio and crystallinity increased with time in OVX group. Elastic modulus and hardness measured by nanoindentation, whole-bone strength measured by biomechanical tests all decreased in OVX group with time. Bone material properties measured by FTIR correlated well with nano or whole-bone level mechanics. However, bone mineral density (BMD), structure, tissue-level and whole-bone mechanical properties did not change with age in sham group. Our study demonstrated that OVX could affect the tissue-level mechanics and bone strength of cortical bone. And this influence was attributed to the time related alterations of mineral and collagen properties, which may help us to design earlier interventions and more effective treatment strategies on osteoporosis.