Pyridinoline Cross-links Research Articles

Abstract 4112931: The Nature of Collagen Cross-links Differs in Reversibly and Irreversibly Infarcted Myocardium

Background: Persistent scarring after myocardial infarction (MI) is a major driver of heart failure in mammals. However, unlike mammals, zebrafish exhibit a remarkable ability to resorb scar, which consists predominantly of collagen in both species. Aims: We aimed here to develop a toolbox of techniques to characterize the nature of collagen in infarcted mammalian and zebrafish hearts. Specifically, we aimed to characterize the early and late cross-links that form in collagen and determine whether differences in these cross-links exist in zebrafish and mammals. Methods: To characterize the initial oxidation and cross-linking of collagen we developed a novel fluorescent probe (TMR-O) that binds to lysine aldehydes on collagen and inform on the degree of collagen cross-linking. Additionally, we used high performance liquid chromatography (HPLC) to quantify degradation-resistant mature collagen cross-links, such as pyridinoline (Pyd), and its precursor, hydroxylysine. Results: TMR-O imaging revealed similar overall CCL levels in both zebrafish and mouse infarcts. However, the irreversible cross-link pyridinoline (Pyd) was abundant in mouse infarcts but completely absent in zebrafish (p<0.0001). High Pyd levels in mice were accompanied by a dynamic increase in lysine hydoxylation (>200%), which was not observed in zebrafish (<10%). Conclusions: While collagen in mammalian and zebrafish infarcts may appear histologically similar, marked biochemical differences exist. Mature degradation-resistant collagen cross-links, such as Pyd form, in murine but not zebrafish infarcts and likely explains why zebrafish can resorb myocardial scar. Targeting Pyd formation in mammals could pave the way for novel therapeutic strategies to promote scar resolution and improve cardiac function after MI.

Urinary PYD/Creatinine Ratio Has Negative Correlation to Serum 25(OH)D and Positive Correlation to Chronic Lead Exposure Index

BACKGROUND: The burden of disease due to lead exposure continues to increase. Lead interferes with 25(OH)D hydroxylation and calcium transport, increasing osteoclastic activity and bone resorption. Pyridinoline crosslinks (PYD), as an indicator of bone damage, can be seen earlier compared to imaging changes. Therefore, it is necessary to determine the correlation between serum 25(OH)D levels and the urinary PYD/creatinine ratio in workers exposed to lead, since up to now, there are only limited studies related to it.METHODS: This cross-sectional study involved 104 workers exposed to lead, selected from parents whose children had blood lead levels above 10 µg/dL. Questionnaires and physical examination were performed to obtain characteristic data from subjects. Data regarding blood lead levels, serum 25(OH)D levels, urinary PYD levels, and urinary creatinine levels were also obtained from various laboratory methods.RESULTS: Most subjects (86.5%) had inadequate serum 25(OH)D. Median blood lead levels was 6.3 (1.2-35.5) µg/dL, chronic lead exposure index was 35.3 (1.2-535.8) years µg/dL, serum 25(OH)D levels was 22 (8-52) ng/mL, and urinary PYD/creatinine ratio was 5.3 (3.6-28.1)×10-6. There was a significant negative correlation between serum 25(OH)D levels and urinary PYD/creatinine ratio in workers exposed to lead. There was also a significant positive correlation between chronic lead exposure index and the urinary PYD/creatinine ratio.CONCLUSION: Since urinary PYD/creatinine ratio is correlated with serum chronic lead exposure index and serum 25(OH)D levels, it suggests that pyridinoline might be a potential biomarker to detect bone metabolism disorder due to the chronic lead exposure. Vitamin D adequacy is also an important factor in preventing bone metabolism disorder amidst chronic lead exposure.KEYWORDS: 25(OH)D, bone resorption, pyridinoline, lead, worker

Changes in the hematobiochemical, acid–base and blood gas elements as well as biomarkers of inflammation and bone metabolism in donkeys (Equus asinus) with acute bleeding

Background: Acute hemorrhage is fatal in equines with a complication of severe hypovolemic shock that causes a sudden death in such cases. Aim: This study was designed to report the influences of acute bleeding in conscious non-sedated donkeys (Equus asinus) on the hematobiochemical variables, acid-base, blood gas elements and markers of inflammation and bone metabolism. Methods: Eight healthy donkeys were used where a total of 900mL of whole blood was collected. Five blood samples were collected from each animal: just before collection of blood (T0); (2) 30min (T1), 60min (T2), 120min (T3) and 240min (T4) later. The blood panels including total white blood cells, lymphocytes, neutrophils, red blood cell counts (RBCs), HCT, hemoglobin (Hg) and RBCs indices were measured. Biochemical parameters and electrolytes were evaluated. The activity of aspartate aminotransferase (AST), creatine kinase (CK) and γ-glutamyl transferase (GGT) were also determined. Complete acid-base and blood gas panel were assessed. Serum amyloid A (SAA), haptoglobin (Hp), osteocalcin (OC), bone alkaline phosphatase (b-ALP) and pyridinoline cross-links (PYD) were measured. Results: The RBCs, Hg and HCT increased significantly at points T1, T2 and T3 compared to T0. The concentrations of total proteins and albumin decreased significantly at points T3 and T4. The blood urea nitrogen concentrations increased significantly at T4. Creatinine concentrations increased significantly at T2 and T3. The AST, GGT and CK decreased significantly. On the other hand, glucose increased significantly at T3 and T4. The pH decreased significantly at points T1, T2, T3 and T4. The PCO2 increased significantly at T 3 and T4. The BE, HCO3 and TCO2 values decreased significantly at T2, T3 and T4. Contrary, the AG increased significantly at points T3 and T4. The potassium increased significantly at T1-T4 and chloride decreased significantly at T3 and T4. Lactate showed significant increases at T1-T4. The SAA, Hp, OC, b-ALP and PYD did not differ significantly at T1-T4. Conclusion: In conscious non-sedated donkeys, induced bleeding resulted in significant changes in the hematobiochemical elements, the acid-base status and blood gas and electrolyte parameters. However, it did not change the markers of inflammation and bone metabolism.

Mmp14 is required for matrisome homeostasis and circadian rhythm in fibroblasts

The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon. Next, we generated tamoxifen-induced Col1a2-Cre-ERT2::Mmp14 KO mice (Mmp14 conditional knockout (CKO)). The CKO mice developed hind limb dorsiflexion and thickened tendons, which accumulated narrow-diameter collagen fibrils causing ultrastructural disorganization. Mass spectrometry of control tendons identified 1195 proteins of which 212 showed time-dependent abundance. In Mmp14 CKO mice 19 proteins had reversed temporal abundance and 176 proteins lost time dependency. Among these, the collagen crosslinking enzymes lysyl oxidase-like 1 (LOXL1) and lysyl hydroxylase 1 (LH1; encoded by Plod2) were elevated and had lost time-dependent regulation. High-pressure chromatography confirmed elevated levels of hydroxylysine aldehyde (pyridinoline) crosslinking of collagen in CKO tendons. As a result, collagen-I was refractory to extraction. We also showed that CRISPR-Cas9 deletion of Mmp14 from cultured fibroblasts resulted in loss of circadian clock rhythmicity of period 2 (PER2), and recombinant MT1-MMP was highly effective at cleaving soluble collagen-I but less effective at cleaving collagen pre-assembled into fibrils. In conclusion, our study shows that circadian clock-regulated Mmp14 controls the rhythmic synthesis of small diameter collagen fibrils, regulates collagen crosslinking, and its absence disrupts the circadian clock and matrisome in tendon fibroblasts.

Tissue anisotropy and collagenomics in porcine penile tunica albuginea: Implications for penile structure-function relationships and tissue engineering.

The tunica albuginea (TA) of the penis is an elastic layer that serves a structural role in penile erection. Disorders affecting the TA cause pain, deformity, and erectile dysfunction. There is a substantial clinical need for engineered replacements of TA, but data are scarce on the material properties and biochemical composition of healthy TA. The objective of this study was to assess tissue organization, protein content, and mechanical properties of porcine TA to establish structure-function relationships and design criteria for tissue engineering efforts. TA was isolated from six pigs and subjected to histomorphometry, quantification of collagen content and pyridinoline crosslinks, bottom-up proteomics, and tensile mechanical testing. Collagen was 20±2%/wet weight (WW) and 53±4%/dry weight (DW). Pyridinoline content was 426 ±131 ng/mg WW, 1011±190 ng/mg DW, and 45±8 mmol/mol hydroxyproline. Bottom-up proteomics identified 14 proteins with an abundance of >0.1% of total protein. The most abundant collagen subtype was type I, representing 95.5±1.5% of the total protein in the samples. Collagen types III, XII, and VI were quantified at 1.7±1.0%, 0.8±0.2%, and 0.4±0.2%, respectively. Tensile testing revealed anisotropy: Young's modulus was significantly higher longitudinally than circumferentially (60±18 MPa vs. 8±5 MPa, p<0.01), as was ultimate tensile strength (16±4 MPa vs. 3±3 MPa, p<0.01). Taken together, the tissue mechanical and compositional data obtained in this study provide important benchmarks for the development of TA biomaterials. STATEMENT OF SIGNIFICANCE: The tunica albuginea of the penis serves an important structural role in physiologic penile erection. This tissue can become damaged by disease or trauma, leading to pain and deformity. Treatment options are limited. Little is known about the precise biochemical composition and biomechanical properties of healthy tunica albuginea. In this study, we characterize the tissue using proteomic analysis and tensile testing to establish design parameters for future tissue engineering efforts. To our knowledge, this is the first study to quantify tissue anisotropy and to use bottom-up proteomics to characterize the composition of penile tunica albuginea.

Innovative workflow for the identification of cathepsin K cleavage sites in type I collagen

Since the late 1990s, cathepsin K cleavage sites in type I collagen have been extensively studied due to its ability to release bone resorption biomarkers such as CTX and NTX. However, gel-based methods and N-sequencing used in these studies lack sensitivity, especially for small to medium peptides. In this work, we propose a degradomics mass spectrometry-based workflow that combines protein digestion, Nano-LC-UDMSE, and several software tools to identify cathepsin K cleavage sites. This workflow not only identified previously known cleavage sites, but also discovered new ones. Multiple cleavage hotspots were found and described in type I α1 and type I α2 collagen, many of which coincided with pyridinoline crosslinks, known to stabilize the triple helix. Our results allowed us to establish a chronology of digestion and conclude that cathepsin K preferentially cleaves the extremities of type I collagen before the helical part. We also found that cathepsin K preferentially cleaves amino acid residues with long and hydrophobic lateral chains at the beginning of digestion, whereas no preferred amino acid residues were identified later in the digestion. In conclusion, our workflow successfully identified new cleavage sites and can be easily applied to other proteins or proteases.

Influences of citrus aurantium juice immersion and resonant vibration on the instrumental properties of different cuts from yak carcasses

The influences of citrus aurantium juice (CAJ) immersion and resonant vibration assisted by the immersion on the instrumental properties, cooking loss, vibrated weight gain of supraspinatus (SS), quadriceps femoris (QF) and longissimus thoracis (LT) muscles from yak carcasses were investigated, as well as the collagen characteristics, pyridinoline cross-links, myofibrillar MFI. In the circumstance of CAJ concentration of 1% and the ratio of meat to liquid 1:2, the shear force of SS and QF were significantly decreased by 20.3% and 20.2% (P < 0.05), and cooking loss significantly decreased by 17% and 23% (P < 0.05), respectively, compared to the control. At this combination of the concentration and the ratio of meat to liquid, the total collagen contents of SS and QF were significantly decreased by 23% and 18%, the collagen heat solubility 1.4 times and 1.5 times that of the raw meat (P < 0.05), respectively. At the pH value closed to the pI of myofibrillar proteins, the immersion assisted resonance with the amplitude of 2 mm and vibration duration of 1 h made the LT shear force to be decreased by 12%, 7.8% and 10.9% than the control, 0.5 h and 1.5 h of vibration duration (P < 0.05), respectively. Furthermore, the ultrastructure in LT gave rise to obvious alteration due to the CAJ immersion and its assisted resonance. The present study has confirmed again that the resonance does cause meat tenderization by means of destroying the integrity of the M-band structure in sarcomere.

Topographical Characterization of the Young, Healthy Human Femoral Medial Condyle.

The medial femoral condyle of the knee exhibits some of the highest incidences of chondral degeneration. However, a dearth of healthy human tissues has rendered it difficult to ascertain whether cartilage in this compartment possesses properties that predispose it to injuries. Assessment of young, healthy tissue would be most representative of the tissue's intrinsic properties. This work examined the topographical differences in tribological, tensile, and compressive properties of young (n = 5, 26.2 ± 5.6 years old), healthy, human medial femoral condyles, obtained from viable allograft specimens. Corresponding to clinical incidences of pathology, it was hypothesized that the lowest mechanical properties would be found in the posterior region of the medial condyle, and that tissue composition would correspond to the established structure-function relationships of cartilage. Young's modulus, ultimate tensile strength, aggregate modulus, and shear modulus in the posterior region were 1.0-, 2.8-, 1.1-, and 1.0-fold less than the values in the anterior region, respectively. Surprisingly, although glycosaminoglycan content is thought to correlate with compressive properties, in this study, the aggregate and shear moduli correlated more robustly to the amount of pyridinoline crosslinks per collagen. Also, the coefficient of friction was anisotropic and ranged 0.22-0.26 throughout the condyle. This work showed that the posteromedial condyle displays lower tensile and compressive properties, which correlate to collagen crosslinks and may play a role in this region's predisposition to injuries. Furthermore, new structure-function relationships may need to be developed to account for the role of collagen crosslinks in compressive properties.

Ion modulatory treatments toward functional self-assembled neocartilage

Signals that recapitulate in vitro the conditions found in vivo, such as hypoxia or mechanical forces, contribute to the generation of tissue-engineered hyaline-like tissues. The cell regulatory processes behind hypoxic and mechanical stimuli rely on ion concentration; iron is required to degrade the hypoxia inducible factor 1a (HIF1α) under normoxia, whereas the initiation of mechanotransduction requires the cytoplasmic increase of calcium concentration. In this work, we propose that ion modulation can be used to improve the biomechanical properties of self-assembled neocartilage constructs derived from rejuvenated expanded minipig rib chondrocytes. The objectives of this work were 1) to determine the effects of iron sequestration on self-assembled neocartilage constructs using two doses of the iron chelator deferoxamine (DFO), and 2) to evaluate the performance of the combined treatment of DFO and ionomycin, a calcium ionophore that triggers cytoplasmic calcium accumulation. This study employed a two-phase approach. In Phase I, constructs treated with a high dose of DFO (100 µM) exhibited an 87% increase in pyridinoline crosslinks, a 57% increase in the Young's modulus, and a 112% increase in the ultimate tensile strength (UTS) of the neotissue. In Phase II, the combined use of both ion modulators resulted in 150% and 176% significant increases in the Young's modulus and UTS of neocartilage constructs, respectively; for the first time, neocartilage constructs achieved a Young's modulus of 11.76±3.29 MPa and UTS of 4.20±1.24 MPa. The results of this work provide evidence that ion modulation can be employed to improve the biomechanical properties in engineered neotissues. Statement of significanceThe translation of tissue-engineered products requires the development of strategies capable of producing biomimetic neotissues in a replicable, controllable, and cost-effective manner. Among other functions, Fe2+ and Ca2+ are involved in the control of the hypoxic response and mechanotransduction, respectively. Both stimuli, hypoxia and mechanical forces, are known to favor chondrogenesis. This study utilized ion modulators to improve the mechanical properties self-assembled neocartilage constructs derived from expanded and rejuvenated costal chondrocytes via Fe2+ sequestration and Ca2+ influx, alone or in combination. The results indicate that ion modulation induced tissue maturation and a significant improvement of the mechanical properties, and holds potential as a tool to mitigate the need for bioreactors and engineer hyaline-like tissues.

The Effects of Acute Blood Loss on Inflammatory and Bone Biomarkers, Acid-base Balance, Blood Gases and Hemato-biochemical Profiles in Sedated Donkeys (Equus asinus)

The current experiment was designed to investigate the possible effects of acute blood loss on the inflammatory and bone biomarkers status, acid-base balance, blood gases, and hemato-biochemical profiles in sedated donkeys (Equus asinus). For the induction of acute blood loss, collected 900mL of blood from the carotid artery of 10 male donkeys sedated with xylazine HCl (1mg/kg). For analysis, five blood samples were collected; collected the 1st (T0) before induction of blood loss and the other 4 (T1, T2, T3 and T4) collected at 30, 60, 120 and 240min post-blood loss. The serum concentrations of amyloid A (SAA) and haptoglobin (Hp) decreased at all-time points after blood loss compared to T0, but the differences were non-significant. The differences in levels of serum biomarkers osteocalcin (OC), β-alkaline phosphatase (β-ALP) and pyridinoline cross-links (PYD) at all-time points post blood loss compared to T0 values were also non-significant. Except for PCO2, the acid-base status and blood gases, including pH, PO2, HCO3, TCO2, sO2, base excess, anion gap and lactate, increased significantly after blood withdrawal. On the contrary, most of the measured hemato-biochemical parameters decreased significantly after blood loss, except for glucose. In conclusion, acute blood loss in sedated donkeys did not influence serum concentrations of the inflammatory biomarkers SAA and Hp and the markers of bone metabolism OC, β-ALP, and PYD. On the contrary, most acid-base, blood gas parameters, and hemato-biochemical parameters differed significantly after blood withdrawal compared to their values before blood loss.

Yucatan Minipig Knee Meniscus Regional Biomechanics and Biochemical Structure Support its Suitability as a Large Animal Model for Translational Research.

Knee meniscus injuries are the most frequent causes of orthopedic surgical procedures in the U.S., motivating tissue engineering attempts and the need for suitable animal models. Despite extensive use in cardiovascular research and the existence of characterization data for the menisci of farm pigs, the farm pig may not be a desirable preclinical model for the meniscus due to rapid weight gain. Minipigs are conducive to in vivo experiments due to their slower growth rate than farm pigs and similarity in weight to humans. However, characterization of minipig knee menisci is lacking. The objective of this study was to extensively characterize structural and functional properties within different regions of both medial and lateral Yucatan minipig knee menisci to inform this model’s suitability as a preclinical model for meniscal therapies. Menisci measured 23.2–24.8 mm in anteroposterior length (33–40 mm for human), 7.7–11.4 mm in width (8.3–14.8 mm for human), and 6.4–8.4 mm in peripheral height (5–7 mm for human). Per wet weight, biochemical evaluation revealed 23.9–31.3% collagen (COL; 22% for human) and 1.20–2.57% glycosaminoglycans (GAG; 0.8% for human). Also, per dry weight, pyridinoline crosslinks (PYR) were 0.12–0.16% (0.12% for human) and, when normalized to collagen content, reached as high as 1.45–1.96 ng/µg. Biomechanical testing revealed circumferential Young’s modulus of 78.4–116.2 MPa (100–300 MPa for human), circumferential ultimate tensile strength (UTS) of 18.2–25.9 MPa (12–18 MPa for human), radial Young’s modulus of 2.5–10.9 MPa (10–30 MPa for human), radial UTS of 2.5–4.2 MPa (1–4 MPa for human), aggregate modulus of 157–287 kPa (100–150 kPa for human), and shear modulus of 91–147 kPa (120 kPa for human). Anisotropy indices ranged from 11.2–49.4 and 6.3–11.2 for tensile stiffness and strength (approximately 10 for human), respectively. Regional differences in mechanical and biochemical properties within the minipig medial meniscus were observed; specifically, GAG, PYR, PYR/COL, radial stiffness, and Young’s modulus anisotropy varied by region. The posterior region of the medial meniscus exhibited the lowest radial stiffness, which is also seen in humans and corresponds to the most prevalent location for meniscal lesions. Overall, similarities between minipig and human menisci support the use of minipigs for meniscus translational research.

Pseudohypoxic HIF pathway activation dysregulates collagen structure-function in human lung fibrosis.

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFβ increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

Meta-analysis of the relationship between collagen characteristics and meat tenderness

Meta-analysis methods were used to investigate the effects of collagen content, solubility and pyridinoline cross-link content on Warner-Bratzler shear force (WBSF) and sensory tenderness in major meat species. Data was collected from the literature on pork, beef and lamb and analyzed independently for each species. The beef data was categorized into subgroups according to muscle (loin and other muscle) and age (young, <18 months and old, ≥18 months). The results showed that in beef, collagen content and pyridinoline cross-link content were positively correlated with WBSF and negatively with sensory tenderness, while collagen solubility was negatively correlated with WBSF. The correlation coefficients were greater in other beef muscles than loin. Significant correlations between collagen content and tenderness attributes were observed in old beef animals. However, non-significant relationships and large variations were found in pork and lamb. More studies with various muscles and ages are required for an in-depth understanding of the relationship between collagen characteristics and meat tenderness.

Increased Collagen Crosslinking in Stiff Clubfoot Tissue: Implications for the Improvement of Therapeutic Strategies.

Congenital clubfoot is a complex musculoskeletal deformity, in which a stiff, contracted tissue forms in the medial part of the foot. Fibrotic changes are associated with increased collagen deposition and lysyl oxidase (LOX)-mediated crosslinking, which impair collagen degradation and increase the tissue stiffness. First, we studied collagen deposition, as well as the expression of collagen and the amount of pyridinoline and deoxypyridinoline crosslinks in the tissue of relapsed clubfoot by immunohistochemistry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA). We then isolated fibroblast-like cells from the contracted tissue to study the potential inhibition of these processes in vitro. We assessed the effects of a LOX inhibitor, β-aminopropionitrile (BAPN), on the cells by a hydroxyproline assay, ELISA, and Second Harmonic Generation imaging. We also evaluated the cell-mediated contraction of extracellular matrix in 3D cell-populated collagen gels. For the first time, we have confirmed significantly increased crosslinking and excessive collagen type I deposition in the clubfoot-contracted tissue. We successfully reduced these processes in vitro in a dose-dependent manner with 10–40 µg/mL of BAPN, and we observed an increasing trend in the inhibition of the cell-mediated contraction of collagen gels. The in vitro inhibitory effects indicate that BAPN has good potential for the treatment of relapsed and resistant clubfeet.

Skeletal muscle progenitors are sensitive to collagen architectural features of fibril size and cross linking.

Muscle stem cells (MuSCs) are essential for the robust regenerative capacity of skeletal muscle. However, in fibrotic environments marked by abundant collagen and altered collagen organization, the regenerative capability of MuSCs is diminished. MuSCs are sensitive to their extracellular matrix environment but their response to collagen architecture is largely unknown. The present study aimed to systematically test the effect of underlying collagen structures on MuSC functions. Collagen hydrogels were engineered with varied architectures: collagen concentration, cross linking, fibril size, and fibril alignment, and the changes were validated with second harmonic generation imaging and rheology. Proliferation and differentiation responses of primary mouse MuSCs and immortal myoblasts (C2C12s) were assessed using EdU assays and immunolabeling skeletal muscle myosin expression, respectively. Changing collagen concentration and the corresponding hydrogel stiffness did not have a significant influence on MuSC proliferation or differentiation. However, MuSC differentiation on atelocollagen gels, which do not form mature pyridinoline cross links, was increased compared with the cross-linked control. In addition, MuSCs and C2C12 myoblasts showed greater differentiation on gels with smaller collagen fibrils. Proliferation rates of C2C12 myoblasts were also higher on gels with smaller collagen fibrils, whereas MuSCs did not show a significant difference. Surprisingly, collagen alignment did not have significant effects on muscle progenitor function. This study demonstrates that MuSCs are capable of sensing their underlying extracellular matrix (ECM) structures and enhancing differentiation on substrates with less collagen cross linking or smaller collagen fibrils. Thus, in fibrotic muscle, targeting cross linking and fibril size rather than collagen expression may more effectively support MuSC-based regeneration.

Silicon supplementation affects mineral metabolism but not bone density or strength in male broilers.

Because leg injuries produce welfare concerns and impact production for broilers, numerous interventions have been suggested as potential solutions. One mineral which may affect bone quality is silicon. The objective of this study was to determine if supplementing bioavailable silicon could affect bone morphology, mineralization, and strength without negatively influencing welfare and meat quality. Male broilers were raised from d 1 after hatching until 42 d of age and randomly assigned to treatment groups for silicon supplementation in water: Control (no supplement, C; n = 125), Normal (0.011 ml supplement/kg bodyweight, N; n = 125) and High (0.063 ml supplement/kg bodyweight, H; n = 125). Toe damage, footpad dermatitis, hock burn, and keel blisters were assessed on d 42. Blood samples were collected from wing veins for serum osteocalcin, pyridinoline cross-links, and mineral analysis. Clinical QCT scans and analysis were conducted immediately before four-point bending tests of tibias. Texture analysis was performed on cooked fillets. Silicon supplementation tended to increase daily water consumption in N and H as compared to C (P = 0.07). Footpad dermatitis and hock burn scores were higher in H than in N or C (P < 0.05 for both comparisons). Supplementation altered serum minerals (P < 0.001), but bone density, morphology, and strength measures were similar among groups. The highest level of supplementation in the current study on a kg bodyweight basis was above recommended intakes but below previous amounts demonstrating silicon’s positive influence on bone, indicating that previously suggested minimum thresholds need to be reevaluated. Factors such as growth rate and mechanical loading likely play a greater role in developing bone quality than trying to supplement on top of good basic nutrition alone.

Physioxia Stimulates Extracellular Matrix Deposition and Increases Mechanical Properties of Human Chondrocyte-Derived Tissue-Engineered Cartilage.

Cartilage tissue has been recalcitrant to tissue engineering approaches. In this study, human chondrocytes were formed into self-assembled cartilage sheets, cultured in physiologic (5%) and atmospheric (20%) oxygen conditions and underwent biochemical, histological and biomechanical analysis at 1- and 2-months. The results indicated that sheets formed at physiological oxygen tension were thicker, contained greater amounts of glycosaminoglycans (GAGs) and type II collagen, and had greater compressive and tensile properties than those cultured in atmospheric oxygen. In all cases, cartilage sheets stained throughout for extracellular matrix components. Type II-IX-XI collagen heteropolymer formed in the neo-cartilage and fibrils were stabilized by trivalent pyridinoline cross-links. Collagen cross-links were not significantly affected by oxygen tension but increased with time in culture. Physiological oxygen tension and longer culture periods both served to increase extracellular matrix components. The foremost correlation was found between compressive stiffness and the GAG to collagen ratio.

The influence of whole-body vibration on heart rate, stride length, and bone mineral content in the mature exercising horse

Research in humans suggests whole-body vibration (WBV) aids in maintaining bone mineral content (BMC) yet results in the horse are less favourable. Anecdotally, WBV is reported to reduce pain and improve performance. This study was designed to test the effect of WBV on exercising horses, hypothesising that WBV would lower heart rate (HR) during treatment, increase BMC, modify markers of bone metabolism, and increase stride length. Eleven horses were randomly assigned into control (CON, n=5) or WBV (VIB, n=6) groups for a 28-day treatment period. Both groups exercised for 1 h, 6 d/wk on a mechanical exerciser. VIB horses received 50 Hz WBV for 45 min, 5 days/wk. Third metacarpal radiographs were taken at 0 and 28 days, and BMC determined via radiographic bone aluminium equivalence (RBAE). Blood samples taken at day 0 and 28 were analysed for serum pyridinoline cross-links (PYD) and osteocalcin (OC). Heart rate was analysed on day 23 for 4 horses per group. Stride length was determined while trotting in hand on day 0 and 28. No influence of WBV on RBAE of any bone cortices, PYD or OC was observed (P&gt;0.10); stride length was also unaffected (P=0.88). A period effect was observed for a decrease in RBAE of the lateral cortex (P=0.01), and a trend towards a decrease was noted in total density (P=0.05), likely an effect of stalling. Compared to baseline, ΔHR declined during treatment (P=0.06) in VIB (-4.8±2.8 bpm) compared to control CON (3.0±2.8 bpm). The results suggest, in normal exercising horses, WBV does not increase BMC, influence markers of bone metabolism, or increase stride length.

Impact microindentation measurements correlate with cortical bone material properties measured by Fourier transform infrared imaging in humans.

Bone Material Strength index (BMSi) measured by Impact Microindentation is generally lower in subjects with fragility fractures independently of BMD values. We recently reported that in humans, BMSi values are strongly associated with material properties of subperiosteal mineralized bone surface (local mineral content, nanoporosity, pyridinoline content). In the present study we investigated the relationship of BMSi with material properties of the whole bone cortex, by analyzing thin sections of iliac crest biopsies (N=12) from patients with different skeletal disorders and a wide range of BMD with or without fractures, by Fourier transform infrared imaging (FTIRI). The calculated parameters were: i) mineral and organic matrix content and their ratio (MM), ii) mineral maturity/crystallinity (MMC) and iii) the ratio of pyridinoline (Pyd) and divalent collagen cross-links (XLR). Results were expressed as images, which were converted to histogram distributions. For each histogram the characteristics recorded were: mean value, mode (most often occurring value), skewness, and kurtosis and their association with BMSi values was examined by correlation analysis. BMSi values were significantly correlated only with MM mean and mode values (r=0.736, p=0.0063, and r=0.855, p=0.0004, respectively), and with XLR mode values (r=-0.632, p=0.0274). The results of the present study demonstrate that BMSi values are strongly associated with MM, a metric that corrects the mineral content for the organic matrix content, and may also depend on organic matrix quality. These and our previous observations strongly suggest that BMSi assesses material properties of cortical bone.

Type I collagen differences in farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand

The integrity and function of Type I collagen (Col-I), a fundamental structural molecule, is central to fish movement. Farmed Chinook salmon in New Zealand are reported to develop a late onset curvature syndrome, lordosis, kyphosis and scoliosis (LKS), associated with inflammation and fibrosis, which affects movement and product quality. To investigate if type I collagen integrity is associated with LKS, salmon from a farm with high LKS (Farm 1) were compared with a farm with low LKS (Farm 2).Representative salmon from Farm 1 and Farm 2 were harvested at 25 months of age and their physical metrics measured. Condition factor (K) was derived. White muscle samples from the abdominal and caudal regions were sampled and analysed. The properties of Col-I were determined using liquid chromatography-electrospray ionization mass spectrometry. The amount of Col-I in white muscle, inferred from hydroxy-proline [Hyp], was 0.071 and 0.130 ([Hyp (mg) / Dry sample (mg)]%) for Farm 1 and Farm 2 respectively. There was a significant (p < .0004) difference (~2-fold) in [Hyp] between farms and significant differences for all crosslinks reported below. Mature crosslinks histidinohydroxymerodesmosine (HHMD) were ~2.5-fold higher in Farm 1 salmon. Immature crosslinks were ~3-fold dihydroxylysinonorleucine (DHLNL) and >4-fold hydroxylysinonorleucine (HLNL) higher in Farm 1 salmon. Mature pyridinoline (PYR) crosslinks were readily detectable in salmon from Farm 2 but below the threshold for reliable detection in those from Farm 1. The mature crosslink of elastin, desmosine (DES), was ~1.5-fold higher in Farm 2 salmon. We have quantified Col-I in the white muscle compartment of farmed Chinook salmon and established methods to compare the crosslink profile. PYR and DHLNL crosslinks associated with myosepta were significantly different between the populations. Salmon from Farm 2 had both a higher proportion of mature PYR crosslinks and higher [Hyp], additive differences that may be of functional significance. Higher levels of crosslinks (HLNL, HHMD) associated with loose connective tissue and the extracellular matrix were seen in salmon from Farm 1 and also associated with condition factor. These results demonstrated differences in the amount of Col-I and crosslink profile of farmed Chinook salmon which could be linked with a population-based susceptibility toward LKS. The association between LKS and genetic and/or husbandry differences requires additional controlled experiments to determine these relationships more precisely.