Collagen type II: From biosynthesis to advanced biomaterials for cartilage engineering.

Collagen type II is the major constituent of cartilage tissue. Yet, cartilage engineering approaches are primarily based on collagen type I devices that are associated with suboptimal functional therapeutic outcomes. Herein, we briefly describe cartilage's development and cellular and extracellular composition and organisation. We also provide an overview of collagen type II biosynthesis and purification protocols from tissues of terrestrial and marine species and recombinant systems. We then advocate the use of collagen type II as a building block in cartilage engineering approaches, based on safety, efficiency and efficacy data that have been derived over the years from numerous in vitro and in vivo studies.

Biophysical and biological characterisation of collagen/resilin-like protein composite fibres.

Collagen type I, in various physical forms, is widely used in tissue engineering and regenerative medicine. To control the mechanical properties and biodegradability of collagen-based devices, exogenous cross-links are introduced into the 3D supramolecular structure. However, potent cross-linking methods are associated with cytotoxicity, whilst mild cross-linking methods are associated with suboptimal mechanical resilience. Herein, we assessed the influence of resilin, a super-elastic and highly stretchable protein found within structures in arthropods where energy storage and long-range elasticity are needed, on the biophysical and biological properties of mildly cross-linked extruded collagen fibres. The addition of resilin-like protein in the 4-arm poly(ethylene glycol) ether tetrasuccinimidyl glutarate cross-linked collagen fibres resulted in a significant increase of stress and strain at break values and a significant decrease of modulus values. The addition of resilin-like protein did not compromise cell metabolic activity and DNA concentration. All groups are supported parallel to the longitudinal fibre axis cell orientation. Herein we provide evidence that the addition of resilin-like protein in mildly cross-linked collagen fibres improves their biomechanical properties, without jeopardising their biological properties.

The past, present and future in scaffold-based tendon treatments.

Tendon injuries represent a significant clinical burden on healthcare systems worldwide. As the human population ages and the life expectancy increases, tendon injuries will become more prevalent, especially among young individuals with long life ahead of them. Advancements in engineering, chemistry and biology have made available an array of three-dimensional scaffold-based intervention strategies, natural or synthetic in origin. Further, functionalisation strategies, based on biophysical, biochemical and biological cues, offer control over cellular functions; localisation and sustained release of therapeutics/biologics; and the ability to positively interact with the host to promote repair and regeneration. Herein, we critically discuss current therapies and emerging technologies that aim to transform tendon treatments in the years to come.

The application of high-pressure treatment in the reduction of phosphate levels in breakfast sausages.

This study investigated effects of high pressure (HP) treatment of pork meat at 150 or 300 MPa for 5 min before manufacturing sausages on the reduction of phosphate levels and compared to sausages manufactured with untreated pork meat (control sausages). Improvement in perceived saltiness, juiciness and overall flavour was observed in sausages manufactured using HP-treated meat at 150 MPa and 0% phosphate, compared to control sausages. Sausages manufactured using meat HP-treated at 150 MPa and 0.25% phosphate (P<0.05) improved hardness of sausages. HP-treated meat at 300 MPa and 0% phosphate decreased juiciness and adhesiveness, while at 0.25% phosphate, adversely affected emulsion stability and sensory attributes. HP treatment did not affect significantly the lightness of the sausages; however, elimination of phosphate reduced (P<0.05) the yellowness, while HP treatment at 150 MPa with 0.25 or 0.5% phosphate increased (P<0.05) redness. HP reatment at 150 MPa has potential for reducing phosphate levels in sausages without significant changes in their functionality and improved acceptability.

Association of polymorphisms in candidate genes with colour, water-holding capacity, and composition traits in bovine M. longissimus and M. semimembranosus.

The objective of this study was to determine the association of single nucleotide polymorphisms (SNP) in selected candidate genes with sensory and technological meat quality traits in commercial cattle. SNP in seven candidate genes were genotyped in 130 crossbred Bos taurus cattle using PCR-RFLP. Reported associations between calpastatin (CAST) and Warner-Bratzler shear force and carboxypeptidase E (CPE) and intra-muscular fat were not confirmed. However, SNP in CAST, amp-activated protein kinase, gamma-3 subunit (PRKAG3), growth hormone receptor (GHR) and stearoyl coA desaturase (SCD) genes were significantly associated with colour traits (p<0.05). The PRKAG3 SNP was additionally associated with cook loss in M. longissimus thoracis et lumborum (p<0.05) and tended towards association in M. semimembranosus (p<0.1). An association with pH was identified for the SCD SNP (p<0.001). The GHR polymorphism was influential on moisture and intra-muscular fat in M. semimembranosus and protein content in both muscles (p<0.05). Only CPE was associated with sensory traits (flavour in M. longissimus, p<0.01).

Association analysis of single nucleotide polymorphisms in DGAT1, TG and FABP4 genes and intramuscular fat in crossbred Bos taurus cattle.

Previous studies have indicated that single nucleotide polymorphisms (SNPs) in the diacylglycerol-O-acyltransferase1 (DGAT1), thyroglobulin (TG) and adipose fatty acid binding protein (FABP4) genes are associated with intramuscular fat (IMF) levels or marbling scores in beef. The objectives were to estimate the frequency of SNPs in these candidate genes in purebred Irish cattle (n=459) and to determine if individual SNPs are associated with IMF values of longissimus thoracis et lumborum (LTL) and semimembranosus (SM) muscle of crossbred animals (n=138). Results indicated no significant association between the SNPs and IMF, despite the power of this study being sufficient to detect an association with SNPs in the DGAT1 and FABP4 genes. The results confirm the lack of an association found by many other studies and suggest that these SNPs are not influential on the divergent IMF levels in the crossbred population tested.

Integrity of nuclear genomic deoxyribonucleic acid in cooked meat: Implications for food traceability.

It is essential to isolate high-quality DNA from muscle tissue for PCR-based applications in traceability of animal origin. We wished to examine the impact of cooking meat to a range of core temperatures on the quality and quantity of subsequently isolated genomic (specifically, nuclear) DNA. Triplicate steak samples were cooked in a water bath (100 degrees C) until their final internal temperature was 75, 80, 85, 90, 95, or 100 degrees C, and DNA was extracted. Deoxyribonucleic acid quantity was significantly reduced in cooked meat samples compared with raw (6.5 vs. 56.6 ng/microL; P < 0.001), but there was no relationship with cooking temperature. Quality (A(260)/A(280), i.e., absorbance at 260 and 280 nm) was also affected by cooking (P < 0.001). For all 3 genes, large PCR amplicons (product size >800 bp) were observed only when using DNA from raw meat and steak cooked to lower core temperatures. Small amplicons (<200 bp) were present for all core temperatures. Cooking meat to high temperatures thus resulted in a reduced overall yield and probable fragmentation of DNA to sizes less than 800 bp. Although nuclear DNA is preferable to mitochondrial DNA for food authentication, it is less abundant, and results suggest that analyses should be designed to use small amplicon sizes for meat cooked to high core temperatures.

Lack of an association between single nucleotide polymorphisms in the bovine leptin gene and intramuscular fat in Bos taurus cattle.

Leptin contributes to the regulation of adiposity and metabolism, and single nucleotide polymorphisms (SNPs) in the leptin gene have been associated with intramuscular fat (IMF) levels in beef. Our objectives were to estimate the frequency of four SNPs in the leptin gene in nine purebred cattle (n=430), to test for linkage disequilibrium and infer haplotypes, and to determine if individual genotypes or estimated haplotypes were associated with IMF values in crossbred cattle (n=244). The four SNP loci were found to be in linkage disequilibrium and thus, the frequencies of each of the 16 possible haplotypes were inferred by maximum likelihood. No significant association between any individual SNP and haplotype was found with the divergent IMF values. Our results suggest that these SNPs are not influential on the divergent IMF levels in the crossbred population tested.

Estimation of factors influencing fatty acid profiles in light lambs.

Fatty acid composition of intramuscular, intermuscular, subcutaneous, omental and kidney knob fat depots of eighty male light lambs (±21kg live weight) from five Spanish sheep breeds was analysed. Fat depot, anatomical depot location (internal, external and intramuscular), breed (Spanish Merino, Grazalema Merino, Churra Lebrijana, Segureña and Montesina), weaning type (weaning at 45 days after birth or no weaning) and subcutaneous fat thickness factors were analyzed using a statistical model to quantify their contribution to the variation of each fatty acid. Production system was the main factor to explain variations in overall fatty acid profiles (34.68%). However, for several fatty acids and indices (arachidonic, linoleic, PUFA, n-3/n-6) anatomical depot location was the most significant factor. Feeding system explained 65.49% of CLA variance, indicating a strong influence of suckling period length on CLA deposition in lambs' fat. Moreover, due to the lack of interaction between anatomical depot location or depot and breed type or weaning system for total CLA, for future research only one depot would be enough to study the effect of those factors on CLA levels.

Thermal stability of connective tissue from porcine muscles.

Connective tissue of three porcine muscles (M. infraspinatus, IS; M. longissimus dorsi, LD; M. semimembranosus, SM) from 27 animals [populations A (n=13, reared in Ireland) and B (n=14, reared in Finland)] was studied by measuring the collagen content, collagen solubility and thermal shrinkage temperature of the connective tissue. Colour and pH were also determined. Collagen solubility was highest in IS (p<0.05) and lowest in SM (p<0.05) although no difference between LD and SM was found in population B. The onset and peak temperatures of thermal shrinkage (T(o) and T(p)) were highest in IS (p<0.05). The lowest T(o) and T(p) were found in SM from population B whereas no differences were seen between LD and SM muscles in population A. It was concluded that the thermal stability of the connective tissue in the three porcine muscles differ. IS, as a dark muscle has high thermal shrinkage temperatures and high collagen solubilities in comparison to the lighter LD and SM muscles which have lower thermal shrinkage temperatures and collagen solubilities. Collagen contents were highest in IS and lowest in LD.

Association of polymorphisms in the calpain I, calpain II and growth hormone genes with tenderness in bovine M. longissimus dorsi.

The objective of this study was to determine if there is an association between tenderness in bovine M. longisimus dorsi (LD) and polymorphisms in the bovine calpain I (exons 9 & 14), calpain II (regulatory subunit) or growth hormone (intron 3) genes. Genomic DNA was isolated from bovine LD (n=281) on which quality attributes (Warner Bratzler shear force (WBSF), sarcomere length and composition) were also characterised. DNA polymorphisms were analysed using polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis. Association analyses were performed between genotypes at the four polymorphic loci and day 14 WBSF values. It was found that the calpain 1 exon 9 genotypes had an association with WBSF such that animals with the GA genotype exhibited decreased WBSF and increased tenderness when compared to animals with the GG genotype (P<0.05). This observation concurs with that of earlier studies, suggesting that this polymorphism is a functional marker for beef tenderness.

Understanding meat quality through the application of genomic and proteomic approaches.

During the past few decades, advances in molecular genetics have led to the identification of multiple genes or genetic markers associated with genes that affect traits of interest in livestock, including single genes of large effect and QTL (genomic regions that affect quantitative traits). Transcriptomics enables analysis of the complete set of RNA transcripts produced by the genome at a given time and provides a dynamic link between the genome, the proteome and the cellular phenotype. Through a functional genomics approach to understanding the molecular basis of meat quality, we can gain further insight into the complex interplay of gene expression events involved in the development of meat quality. Proteomics permits visualisation of the protein content of the cell under varying conditions, combining powerful separation techniques with highly sensitive analytical mass spectrometry. To date, both the human and bovine genome projects have advanced our understanding of gene expression and helped elucidate the function of large portions of the genome. Advantages from this research have permeated through to a broader spectrum of research including that of meat science. Meat quality is manifested through a complexity of events in the muscle and their interactions with many environmental stimuli in both the live animal and during the post-mortem period. A lot of progress has been made in our understanding of the biological processes that contribute to the delivery of consistent quality meat. Through the application of tools of genomics and proteomics we are gaining a deeper insight into these processes and their interaction with environmental factors. Knowledge gained from these approaches can be beneficial in defining and optimising management systems for quality, providing assurance of meat quality and in tailoring quality to suit market needs.

The influence of biochemical differences on the variation in tenderness of M. longissimus dorsi of Belgian Blue steers managed homogenously pre and post-slaughter.

The objective of this experiment was to determine the contribution of some biochemical processes of postmortem muscle to the variation in tenderness of beef from Belgian Blue bull cross Holstein-Friesian steers (n=10). These animals were managed optimally from conception to consumption with the aim of reducing tenderness variation. The M. longissimus dorsi (LD) from the left hand side (LHS) and the right hand side (RHS) were analysed for variation in tenderness using Bartletts test. The quality measurements included pH, temperature, Warner Bratzler shear force, sensory tenderness, chemical composition and sarcomere length. Biochemical measurements included myofibrilar proteolysis, glycolytic potential, adenine/inosine ratio and collagen content. No difference for variances or means were observed between LHS and RHS for chemical, quality or biochemical attributes. Biochemical variation was greater than the variation observed in most of the quality attributes measured. Proteolysis was the main biochemical contributor to the variation in shear force tenderness after 2 and 7 as postmortem, but not sensory tenderness. Glycolysis levels and adenine/inosine ratio explained much of the variation in sensory tenderness, but not WBSF. Collagen content in the LD muscles did not explain variation in shear force or sensory tenderness. This would suggest biochemical variation is one of the main contributors to variation in tenderness of beef managed optimally pre- and post-slaughter.

Determination of potential inherent variability when measuring beef quality.

Probes, which can be used on-line to rapidly and efficiently detect beef quality attributes (colour and tenderness), are currently being considered to predict ultimate beef quality. The contribution of the inherent sampling variability (due to factors such as muscle location) needs to be evaluated in order to optimise the sampling procedure for these measurements. The main objective of this trial was to monitor some sampling factors which may contribute to variation in pH and various quality attributes in bovine M. longissimus thoracis et lumborum (LTL). Location along the muscle did not impact on colour measurements (P⩾0.05). Location had no effect on cook loss, Warner Bratzler shear force (WBSF) and sarcomere length values (P⩾0.05). The moisture and intramuscular fat contents did vary (P⩾0.05); however, as the differences were very small this may not be of practical significance. Different models of pH meters/probes had a significant influence (P⩽0.01) on recorded pH values. However pH did not differ significantly (P⩾0.05) along the length of the LTL. The type of meter used also had a significant impact on colour readings and 1 h 'blooming' time was observed as optimal for measuring colour on beef.

Decreasing variation in the eating quality of beef through homogenous pre- and post-slaughter management.

The objective of this experiment was to determine the effectiveness of current "best-practise" management of steers pre- and post-slaughter in reducing variation in the eating quality of beef. Steers sired by one Belgian Blue bull from Holstein-Friesian cows were managed optimally from birth to slaughter. Animals were slaughtered at target body weights and subcutaneous fat scores of 620 kg and 4L (LH) (n=23) or 720 kg and 4H (HH) (n=24). On each slaughter occasion, commercial steers with similar carcass weights and classification scores to the homogenous steers were selected from the factory lairage; n=19 for light commercial steers (LC) and n=20 for heavy commercial steers (HC). Carcasses were hung by the pubic bone at 10 °C for 10 h and 2 °C until 24 h postmortem, when M. longissimus dorsi and M. semimembranosus muscles were excised. Following ageing for 2, 7 and/or 14 days postmortem, eating quality was assessed. Muscle from HH steers was more variable in terms of tenderness, protein, moisture and water-holding capacity compared to muscle from LH steers within LD muscle. Muscle from HC steers was more variable in terms of tenderness, redness colour, protein and intramuscular fat compared to muscle from LC steers within LD muscle. Applying best practice management to the homogenous and commercial steers in the present experiment reduced variances in Warner Bratzler shear force (25.69 and 23.9, respectively) compared to variance (154.9) of previous research carried out by the present authors.

Colour, composition and eating quality of beef from the progeny of two Charolais sires.

Eating quality and variation within eating quality attributes of beef from young bull progeny of a Charolais sire of average conformation heritability (CF44) (n=14) and young bull progeny of a Charolais sire of good conformation heritability (IC27) (n=16) were examined. The M. longissimus dorsi (up to 12th and/or 13th ribs) was excised 24 h post-slaughter and eating quality attributes analysed at 2, 7 and 14 days postmortem. While progeny muscularity and carcass weight reflected that of each sire, in general no variation was observed in the quality attributes. In addition no significant difference in mean values was evident between sire progenies for carcass and meat quality attributes examined. Significant variation was observed in colour after 2 days ageing, but this was not evident after 7 or 14 days ageing. Average sarcomere length did differ significantly (p<0.05) between progeny of both sire types (CF44=1.87 µm and IC27=1.77 µm), but did not appear to impact on tenderness. The similarity between the progeny of the average or good conformation sires examined in this experiment suggests such sires have no effect on the eating quality of their young bull beef progeny.

Quantifying the extent of variation in the eating quality traits of the M. longissimus dorsi and M. semimembranosus of conventionally processed Irish beef.

The aim of this investigation was to quantify the scale of variation in the eating quality of two commercial beef muscles, M. longissimus dorsi (LD) and M. semimembranosus (Sm). Both the LD and Sm were excised from steers (n=81) and heifers (n=81) (classification grade O4H, O4L, R4H, R4L) within 48 h postmortem, vacuum packaged and stored at 4 °C until tested for eating quality at 14 days postmortem. Quality measurements analysed were: pH, Warner Bratzler shear force, sensory attributes, sarcomere length, Hunter L a b muscle and subcutaneous fat colour and chemical composition. Extent of variation in many eating quality measurements, with the exception of most sensory attributes and subcutaneous fat colour, depended on gender, classification grade or a combination of both. The LD was more variable than the Sm for most quality attributes and heifers were more variable than steers. No one carcass grade was more variable over all attributes analysed; with different grades causing higher or lower variances within certain attributes. Knowledge of the current scale of variation in the eating quality of beef is required by the meat industry, and is one that requires further research.

Evaluation of rib steak colour from Friesian, Hereford and Charolais heifers pastured or overwintered prior to slaughter.

Heifers (n=10) were randomly selected from the slaughter line of a local factory each month for a period of 21 months. Rib steak (sampled at the 10th rib) from the left side of each carcass was taken for analysis. The cattle breeds selected during this study were Friesian, Hereford and Charolais. The mean weight of the left side for all carcasses was 146.6 (S.E.M.= 1.0kg). Graded carcasses selected for sampling during this trial were classified using the EUROP scale and the specific heifer grades chosen were factory grades EO4L and EO4H. Initial Hunter 'a' values (on the day of arrival in the laboratory) of rib steak from heifers finished between November and March (overwintered) were significantly (P<0.001) higher than Hunter 'a' values from heifers finished between April and October (pastured). After storage at 4 °C under simulated retail display conditions for 6 days, the Hunter 'a' values for overwintered samples were also significantly (P<0.001) greater than those for pastured samples. Breed also had an effect on the colour of the meat. After storage for 6 days, Hunter 'a' values of rib steak from Charolais were significantly (P<0.05) higher than either Friesian or Hereford. Pastured heifers had significantly (P<0.05) higher levels of the monounsaturated fatty acid C16.1 in the total lipid fraction of rib steak (neutral and polar) than samples taken from overwintered heifers. Pastured heifers had significantly (P<0.01) higher levels of the polyunsaturated fatty acid (PUFA) C18.3 in the phospholipid fraction than those from overwintered cattle. However, Hereford had significantly (P<0.05) higher levels of C14.0, C16.1 and C18.0 in the phospholipid fraction than those found in Friesian and Charolais. The level of α-tocopherol in the muscle was not affected by either pasturing/overwintering or breed. However, Continental breeds had significantly (P<0.05) higher levels of α-tocopherol in adipose tissue than Friesian.

Plasma OLC is elevated in mild experimental uremia but is not associated with hypertension.

BACKGROUND: Little is known about the renal handling of endogenous ouabain-like compound (OLC). The aim of this study was to determine the normal renal clearance of OLC and the effect of mild experimental uremia on plasma OLC and its clearance. METHODS: Male Wistar rats were studied 8 weeks after subtotal (5/6th) nephrectomy (n = 8) and compared with a control sham-operated group (n = 8). RESULTS: Plasma creatinine and OLC were higher in uremic animals compared with controls (creatinine 76+/-5.6 micromol/L v 45+/-9.6 micromol/L, respectively, P < .00005; OLC 195+/-62 pmol/L v 121+/-62 pmol/L, P < .02). Creatinine clearance and OLC clearance were lower in uremic animals compared with controls (creatinine 1.06+/-0.12 mL/min v 1.58+/-0.32 mL/min, respectively, P < .002; OLC 23.6+/-10.4 microL/min v 33.2+/-11.4 microL/min, P < .05). There were no significant differences (all P > .05) between the uremic and control groups in the fractional clearance of OLC (uremic 2.3%+/-1.0% v control 2.2%+/-1.0%), OLC excretion rate (uremic 6.2+/-2.4 pmol/24 h v control 5.0+/-1.1 pmol/24 h) or in the mean systolic blood pressure (BP) (uremic 132+/-13 mm Hg v control 126+/-3 mm Hg). The amount of OLC excreted per unit of functioning nephron mass was 78% higher in uremic animals than in controls. The rate of tubular absorption varied linearly with filtered load, did not differ between groups, and showed no evidence of saturation. CONCLUSIONS: The kidneys are an important excretion route for plasma OLC and moderate but significant increases may occur without inducing hypertension in the short term. The low fractional clearance of OLC is most likely due to tubular absorption and/or catabolism.

Phenotypic properties and characteristics of superoxide production by mouse coronary microvascular endothelial cells.

Coronary microvascular endothelial cells exert (patho)physiological effects on the function of cardiac myocytes, which may be studied experimentally using pure cell populations. As an essential pre-requisite to the investigation of cells from gene-modified mice, we studied the phenotypic properties of coronary microvascular endothelial cells isolated from normal mice, and biochemically characterized the superoxide production by these cells. Microvascular endothelial cells were isolated from devitalized mouse ventricular tissue after sequential digestion with collagenase, trypsin and DNase. Coronary microvascular endothelial cells were separated from cardiac myocytes and other cells by differential centrifugation, plating and culture. Mouse coronary microvascular endothelial cells showed an irregular "cobblestone" morphology at confluence, were >98% positive for CD31 by FACS analysis, and were also positive for VE-cadherin and endothelial-type nitric oxide synthase (eNOS) by confocal microscopy. The cells took up fluorescently labelled, acetylated low-density lipoprotein, but were negative for a alpha -smooth muscle actin, desmin and cytokeratin. Unlike human endothelial cells, mouse coronary microvascular endothelial cells only weakly expressed von Willebrand factor. Immunoblotting showed that the mouse cells expressed components of a phagocyte-type NADPH oxidase. They exhibited NADPH-dependent O(2)(-)-generating activity, which was increased by angiotensin II but completely inhibited by diphenyleneiodonium. Thus, mouse coronary microvascular endothelial cells express both eNOS and NADPH oxidase, interactions between which may play a role in endothelial cell pathophysiology.