Glutaraldehyde as a crosslinking agent for collagen-based biomaterials

The formation of Schiff bases during crosslinking of dermal sheep collagen (DSC) with glutaraldehyde (GA), their stability and their reactivity towards GA was studied. All available free amine groups had reacted with GA to form a Schiff base within 5 min after the start of the reaction under the conditions studied (0.5% (w/w) GA). Before crosslinks are formed the hydrolysable Schiff bases initially present were stabilized by further reaction with GA molecules. An increase in shrinkage temperature (T _s) from 56°C for non-crosslinked DSC (N-DSC) to 78°C for GA crosslinked DSC (G-DSC) was achieved after crosslinking for 1 h. From the relationship between the free amine group content and the T _s during crosslinking it was concluded that higher GA concentrations and longer reaction times will result in the introduction of pendant-GA-related molecules rather than crosslinks. After 24 h crosslinking an average uptake of 3 GA molecules per reacted amine group was found. No increase in the tensile strength of the materials was observed after crosslinking, which may be a result of formation of crosslinks within the fibres rather than in between fibres. Aligning of the fibres by applying a pre-strain to the samples and subsequent crosslinking yielded materials with an increased tensile strength.     

The role of taut tie molecules on the mechanical properties of gel-spun UHMWPE fibres

Summary Gel-spun hot-drawn UHMWPE fibres are known to display a high tensile modulus and strength. These properties are treated in a simple morphological model, explaining the approach of nearly the crystal modulus for fibres attaining only a fraction of the theoretical tensile strength and elongation at break.Creep experiments on irradiated gel-spun UHMWPE fibres serve as an additional test for the application of the model.     

Estimating the energetic cost of feeding excess dietary nitrogen to dairy cows

Feeding N in excess of requirement could require the use of additional energy to metabolize excess protein, and to synthesize and excrete urea; however, the amount and fate of this energy is unknown. Little progress has been made on this topic in recent decades, so an extension of work published in 1970 was conducted to quantify the effect of excess N on ruminant energetics. In part 1 of this study, the results of previous work were replicated using a simple linear regression to estimate the effect of excess N on energy balance. In part 2, mixed model methodology and a larger data set were used to improve upon the previously reported linear regression methods. In part 3, heat production, retained energy, and milk energy replaced the composite energy balance variable previously proposed as the dependent variable to narrow the effect of excess N. In addition, rumen degradable and undegradable protein intakes were estimated using table values and included as covariates in part 3. Excess N had opposite and approximately equal effects on heat production (+4.1 to +7.6 kcal/g of excess N) and retained energy (?4.2 to ?6.6 kcal/g of excess N) but had a larger negative effect on milk gross energy (?52 to ?68 kcal/g of excess N). The results suggest that feeding excess N increases heat production, but more investigation is required to determine why excess N has such a large effect on milk gross energy production.     

Heparinized hydroxyapatite/collagen three-dimensional scaffolds for tissue engineering

Currently, in bone tissue engineering research, the development of appropriate biomaterials for the regeneration of bony tissues is a major concern. Bone tissue is composed of a structural protein, collagen type I, on which calcium phosphate crystals are enclosed. For tissue engineering, one of the most applied strategies consists on the development and application of three dimensional porous scaffolds with similar composition to the bone. In this way, they can provide a physical support for cell attachment, proliferation, nutrient transport and new bone tissue infiltration. Hydroxyapatite is a calcium phosphate with a similar composition of bone and widely applied in several medical/dentistry fields. Therefore, in this study, hydroxyapatite three dimensional porous scaffolds were produced using the polymer replication method. Next, the porous scaffolds were homogeneously coated with a film of collagen type I by applying vacuum force. Yet, due to collagen degradability properties, it was necessary to perform an adequate crosslinking method. As a result, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was employed as an efficient and non-toxic crosslinking method in this research. The composites were characterized by means of SEM, DSC and TNBS. Furthermore, heparin was incorporated in order to accomplish sustained delivery of a growth factor of interest namely, bone morphogenetic proteins (BMP-2). BMP-2 binding and release of non-heparinized and heparinized scaffolds was evaluated at specific time points. The incorporation of heparin leads to a reduced initial burst phase when compared to the non heparinized materials. The results show a beneficial effect with the incorporation of heparin and its potential as a localized drug delivery system for the sustained release of growth factors.     

Short communication: Relationship between lysine/methionine ratios and glucose levels and their effects on casein synthesis via activation of the mechanistic target of rapamycin signaling pathway in bovine mammary epithelial cells

The synthesis of protein requires the availability of specific AA and a large supply of energy in bovine mammary epithelial cells (BMEC). Whether an interaction exists between Lys/Met ratio and glucose level on milk protein synthesis and its potential regulatory mechanism is unclear. We investigated the effects of different Lys/Met ratios and glucose levels on casein synthesis-related gene expression in BMEC to elucidate the underlying molecular mechanisms. Primary BMEC were subjected to 4 treatments for 36 h, arranged in a 2 × 2 factorial design with Lys/Met ratios of 3:1 (1.2:0.4 mM, LM3.0; total AA = 8.24 mM) and 2.3:1 (1.4:0.6 mM, LM2.3; total AA = 8.64 mM) and glucose levels of 17.5 mM (high glucose level) and 2.5 mM (low glucose level). No interactions between Lys/Met ratio and glucose level on cell viability, cell cycle progression, mRNA, or protein expression levels were found. High glucose level increased cell proliferation and promoted cell cycle transition from intermediate phase (G1 phase) to synthesis (S phase) by approximately 50%, whereas Lys/Met ratio had no effect. Both mRNA and protein abundance of α_S1-casein and β-casein were positively affected by LM3.0, whereas a high glucose level increased protein abundance of α_S1-casein and β-casein and increased gene expression of CSN1S1 but not of CSN2. Furthermore, high glucose increased the mRNA abundance of ELF5 and decreased that of GLUT8, enhanced protein expression of total and phosphorylated mechanistic target of rapamycin (mTOR), and decreased phosphorylated AMP-activated protein kinase (AMPK) levels. Treatment LM3.0 had a stimulatory effect on total and phosphorylated mTOR but did not affect AMPK phosphorylation. The mRNA levels of JAK2, ELF5, and RPS6KB1 were upregulated and mRNA levels of EIF4EBP1 were downregulated with LM3.0 compared with LM2.3. Our results indicate that casein synthesis was regulated by Lys/Met ratio via JAK2/ELF5, mTOR, and its downstream RPS6KB1 and EIF4EBP1 signaling. In contrast, glucose regulated casein synthesis through promoting cell proliferation, accelerating cell cycle progression, and activating the ELF5 and AMPK/mTOR signaling pathways. Within the range of substrate levels in the present study, a change in Lys/Met ratio had a stronger effect on abundance of α_S1-casein and β-casein than a change in glucose level.     

Effects of forage type, forage to concentrate ratio, and crushed linseed supplementation on milk fatty acid profile in lactating dairy cows

The effects of an increasing proportion of crushed linseed (CL) in combination with varying forage type (grass or corn silage) and forage to concentrate ratio (F:C), and their interactions on milk fatty acid (FA) profile of high-producing dairy cows was studied using a 3-factor Box-Behnken design. Sixteen Holstein and 20 Swedish Red cows were blocked according to breed, parity, and milk yield, and randomly assigned to 4 groups. Groups were fed different treatment diets formulated from combinations of the 3 main factors each containing 3 levels. Forage type (fraction of total forage dry matter, DM) included 20, 50, and 80% grass silage, with the remainder being corn silage. The F:C (DM basis) were 35:65, 50:50, and 65:35, and CL was supplied at 1, 3, and 5% of diet DM. Starch and neutral detergent fiber content (DM basis) of the treatment diets ranged from 117 to 209 g/kg and 311 to 388 g/kg, respectively. Thirteen treatment diets were formulated according to the Box-Behnken design. During 4 experimental periods of 21 d each, all treatment diets were fed, including a repetition of the center point treatment (50% grass silage, 50:50 F:C, 3% CL) during every period. Intake, production performance, and milk FA profile were measured, and response surface equations were derived for these variables. Shifting from 80% grass silage to 80% corn silage in the diet linearly increased dry matter intake (DMI), net energy for lactation (NE_L) intake, cis-9,cis-12-C18:2 (C18:2n-6) intake, and milk yield, and linearly decreased cis-9,cis-12,cis-15-C18:3 (C18:3n-3) intake and milk fat content. Shifting from a high forage to a high concentrate diet linearly increased DMI, NE_L intake, C18:2n-6 intake, and milk yield, and decreased milk fat content. Supplementation of CL linearly increased C18:3n-3 intake, but had no effect on DMI, NE_L intake, milk yield, or milk fat content. Shifting from 80% grass silage to 80% corn silage linearly increased proportions of trans-10-C18:1 and C18:2n-6 in milk fat, whereas the proportions of trans-11,cis-15-C18:2 and C18:3n-3 linearly decreased. Significant interactions between CL supplementation and F:C were found for proportions of trans-10-C18:1, trans-15-C18:1, cis-15-C18:1, trans-11,cis-15-C18:2, and C18:3n-3 in milk fat, with the highest levels achieved when the diet contained 5% CL and a 35:65 F:C ratio. The effect of supplementing CL on several milk FA proportions, including C18:2n-6 and C18:3n-3, depends significantly on the F:C ratio and forage type in the basal diet.     

Effects of feeding rapeseed oil, soybean oil, or linseed oil on stearoyl-CoA desaturase expression in the mammary gland of dairy cows

Stearoyl-CoA desaturase (SCD) is an important enzyme in the bovine mammary gland, and it introduces a double bond at the Δ⁹ location of primarily myristoyl-, palmitoyl-, and stearoyl-CoA. The main objective of this study was to compare the effects of various fatty acids (FA) typically present in dairy cow rations on the expression of SCD1 and SCD5 in the mammary gland of dairy cows. Twenty-eight Holstein-Friesian cows were randomly assigned to 1 of 4 dietary treatments. The dietary treatments were a basal diet supplemented (dry matter basis) with 2.7% rapeseed oil as a source of C18:1 cis-9; 2.7% soybean oil as a source of C18:2 cis-9,12; 2.7% linseed oil as a source of C18:3 cis-9,12,15; or 2.7% of a 1:1:1 mixture of the 3 oils. The oil supplements were included in the concentrate, which was fed together with corn silage and grass silage. In addition, cows were grazing on pasture, consisting mainly of perennial ryegrass, during the day. Biopsies from the mammary gland were taken and analyzed for mRNA expression of SCD1 and SCD5 by using quantitative real-time PCR. Milk yield as well as milk protein and fat contents did not differ among the 4 dietary treatments. Dietary supplementation with rapeseed oil and linseed oil increased proportions of C18:1 cis-9 and C18:3 cis-9,12,15 in blood plasma, respectively, compared with the other treatments. Supplementation with soybean oil and linseed oil increased milk FA proportions of C18:2 cis-9,12 and C18:3 cis-9,12,15, respectively, but supplementation with rapeseed oil did not increase C18:1 cis-9 in milk. Mammary SCD1 expression was reduced by supplementation of soybean oil compared with rapeseed oil and linseed oil. In contrast, SCD5 expression did not differ among the 4 treatments. The C16 and C18 desaturation indices, representing proxies for SCD activity, were lower for the soybean oil diet compared with the diet supplemented with a mixture of the 3 oils. In conclusion, our study shows that mammary SCD1 expression is significantly downregulated in dairy cows by feeding unprotected soybean oil compared with rapeseed oil or linseed oil, and this is partially reflected by the lower desaturase indices in the milk. Furthermore, mammary SCD5 expression appears to be differently regulated than expression of SCD1.     

Cost-effectiveness of feeding strategies to reduce greenhouse gas emissions from dairy farming

The objective of this paper was to evaluate the cost-effectiveness of 3 feeding strategies to reduce enteric CH₄ production in dairy cows by calculating the effect on labor income at the farm level and on greenhouse gas (GHG) emissions at the chain level (i.e., from production of farm inputs to the farm gate). Strategies included were (1) dietary supplementation of an extruded linseed product (56% linseed; 1 kg/cow per day in summer and 2 kg/cow per day in winter), (2) dietary supplementation of a nitrate source (75% nitrate; 1% of dry matter intake), and (3) reducing the maturity stage of grass and grass silage (grazing at 1,400 instead of 1,700 kg of dry matter/ha and harvesting at 3,000 instead of 3,500 kg of dry matter/ha). A dairy farm linear programing model was used to define an average Dutch dairy farm on sandy soil without a predefined feeding strategy (reference situation). Subsequently, 1 of the 3 feeding strategies was implemented and the model was optimized again to determine the new economically optimal farm situation. Enteric CH₄ production in the reference situation and after implementing the strategies was calculated based on a mechanistic model for enteric CH₄ and empirical formulas explaining the effect of fat and nitrate supplementation on enteric CH₄ production. Other GHG emissions along the chain were calculated using life cycle assessment. Total GHG emissions in the reference situation added up to 840 kg of CO₂ equivalents (CO₂e) per t of fat- and protein-corrected milk (FPCM) and yearly labor income of €42,605. Supplementation of the extruded linseed product reduced emissions by 9 kg of CO₂e/t of FPCM and labor income by €16,041; supplementation of the dietary nitrate source reduced emissions by 32 kg of CO₂e/t of FPCM and labor income by €5,463; reducing the maturity stage of grass and grass silage reduced emissions by 11 kg of CO₂e/t of FPCM and labor income by €463. Of the 3 strategies, reducing grass maturity was the most cost-effective (€57/t of CO₂e compared with €241/t of CO₂e for nitrate supplementation and €2,594/t of CO₂e for linseed supplementation) and had the greatest potential to be used in practice because the additional costs were low.