Chemical modification of extracellular matrix by cold atmospheric plasma-generated reactive species affects chondrogenesis and bone formation.

The goal of this study was to investigate whether cold plasma generated by dielectric barrier discharge (DBD) modifies extracellular matrices (ECM) to influence chondrogenesis and endochondral ossification. Replacement of cartilage by bone during endochondral ossification is essential in fetal skeletal development, bone growth and fracture healing. Regulation of this process by the ECM occurs through matrix remodelling, involving a variety of cell attachment molecules and growth factors, which influence cell morphology and protein expression. The commercially available ECM, Matrigel, was treated with microsecond or nanosecond pulsed (μsp or nsp, respectively) DBD frequencies conditions at the equivalent frequencies (1 kHz) or power (~1 W). Recombinant human bone morphogenetic protein-2 was added and the mixture subcutaneously injected into mice to simulate ectopic endochondral ossification. Two weeks later, the masses were extracted and analysed by microcomputed tomography. A significant increase in bone formation was observed in Matrigel treated with μsp DBD compared with control, while a significant decrease in bone formation was observed for both nsp treatments. Histological and immunohistochemical analysis showed Matrigel treated with μsp plasma increased the number of invading cells, the amount of vascular endothelial growth factor and chondrogenesis while the opposite was true for Matrigel treated with nsp plasma. In support of the in vivo Matrigel study, 10 T1/2 cells cultured in vitro on μsp DBD-treated type I collagen showed increased expression of adhesion proteins and activation of survival pathways, which decreased with nsp plasma treatments. These results indicate DBD modification of ECM can influence cellular behaviours to accelerate or inhibit chondrogenesis and endochondral ossification. Copyright © 2016 John Wiley & Sons, Ltd

Applications of Fourier Transform Infrared (FT-IR) Microscopy to the Study of Mineralization in Bone and Cartilage

Knowledge of the phase, composition, and crystallite size and perfection of the mineral in normal and abnormally calcified tissues provides insight into the mechanism by which this mineral was deposited. These data also can be used to develop rational therapies for pathological conditions characterized by abnormal mineral deposition. As illustrated in this review, coupling of an optical microscope with a Fourier transform infrared (FT-IR) spectrophotometer permits the mapping at 20 μm spatial resolution of changes in mineral characteristics (content, particle size, composition) in the growth plate, in bone biopsies, in mineralizing cell culture systems, and in soft tissue calcifications. Based on the infrared properties of apatitic compounds, and comparisons with x-ray diffraction data, correlations have been established from which mineral parameters can be determined. The validity of these spectral correlations has been demonstrated by independent measurements of mineral content (ash weight), and crystal particle size (dark field electron microscopy)

Unusual Fragmentation Pathways in Collagen Glycopeptides

Collagens are the most abundant glycoproteins in the body. One characteristic of this protein family is that the amino acid sequence consists of repeats of three amino acids –(X—Y—Gly)n. Within this motif, the Y residue is often 4-hydroxyproline (HyP) or 5-hydroxylysine (HyK). Glycosylation in collagen occurs at the 5-OH group in HyK in the form of two glycosides, galactosylhydroxylysine (Gal-HyK) and glucosyl galactosylhydroxylysine (GlcGal-HyK). In collision induced dissociation (CID), collagen tryptic glycopeptides exhibit unexpected gas-phase dissociation behavior compared to typical N- and O-linked glycopeptides, i.e. in addition to glycosidic bond cleavages, extensive cleavages of the amide bonds are observed. The Gal- or GlcGal- glycan modifications are largely retained on the fragment ions. These features enable unambiguous determination of the amino acid sequence of collagen glycopeptides and the location of the glycosylation site. This dissociation pattern was consistent for all analyzed collagen glycopeptides, regardless of their length or amino acid composition, collagen type or tissue. The two fragmentation pathways – amide bond and glycosidic bond cleavage – are highly competitive in collagen tryptic glycopeptides. The number of ionizing protons relative to the number of basic sites (i.e. Arg, Lys, HyK and N-terminus) is a major driving force of the fragmentation. We present here our experimental results and employ quantum mechanics calculations, to understand the factors enhancing the labile character of the amide bonds and the stability of hydroxylysine glycosides in gas phase dissociation of collagen glycopeptides

Glycosylation and Cross-linking in Bone Type I Collagen

Fibrillar type I collagen is the major organic component in bone, providing a stable template for mineralization. During collagen biosynthesis, specific hydroxylysine residues become glycosylated in the form of galactosyl- and glucosylgalactosyl-hydroxylysine. Furthermore, key glycosylated hydroxylysine residues, α1/2-87, are involved in covalent intermolecular cross-linking. Although cross-linking is crucial for the stability and mineralization of collagen, the biological function of glycosylation in cross-linking is not well understood. In this study, we quantitatively characterized glycosylation of non-cross-linked and cross-linked peptides by biochemical and nanoscale liquid chromatography-high resolution tandem mass spectrometric analyses. The results showed that glycosylation of non-cross-linked hydroxylysine is different from that involved in cross-linking. Among the cross-linked species involving α1/2-87, divalent cross-links were glycosylated with both mono- and disaccharides, whereas the mature, trivalent cross-links were primarily monoglycosylated. Markedly diminished diglycosylation in trivalent cross-links at this locus was also confirmed in type II collagen. The data, together with our recent report (Sricholpech, M., Perdivara, I., Yokoyama, M., Nagaoka, H., Terajima, M., Tomer, K. B., and Yamauchi, M. (2012) Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance. J. Biol. Chem. 287, 22998–23009), indicate that the extent and pattern of glycosylation may regulate cross-link maturation in fibrillar collagen

Temporal Changes in Collagen Cross-Links in Spontaneous Articular Cartilage Repair

Objective: Little is known about how the biochemical properties of collagen change during tissue regeneration following cartilage damage. In the current study, temporal changes in cartilage repair tissue biochemistry were assessed in a rabbit osteochondral defect. Design: Bilateral full-thickness 3-mm osteochondral trochlear groove defects were created in 54 adult male skeletally mature New Zealand white rabbits, and tissue repair was monitored over 16 weeks. Collagen content, cross-links, lysyl hydroxylation, gene expression, histological grading, and Fourier transform infrared analyses were performed at 2, 4, 6, 8, 12, and 16 weeks. Results: Defect fill occurred at ~4 weeks postinjury; however, histological grading showed that the repair tissue never became normal, primarily due to the presence of fibrocartilage. Gene expression levels of Col1a1 and Col IIaI were higher in the defect compared with adjacent regions. Collagen content in the repair tissue reached the level of normal cartilage at 6 weeks, but it took 12 weeks for the extent of lysine hydroxylation to return to normal. Divalent immature cross-links markedly increased in the early stages of repair. Though the levels gradually diminished thereafter, they never returned to the normal levels. The mature cross-link, pyridinoline, gradually increased with time and nearly reached normal levels by week 16. Infrared imaging data of protein content paralleled the biochemical data. However, collagen maturity, a parameter previously shown to reflect collagen cross-link ratios in bone, did not correlate with the biochemical determination of cross-links in the repair tissue. Conclusion: Collagen biochemical data could provide markers for clinical monitoring in a healing defect

Monitoring the Progression of Spontaneous Articular Cartilage Healing with Infrared Spectroscopy

Objective. Evaluation of early compositional changes in healing articular cartilage is critical for understanding tissue repair and for therapeutic decision-making. Fourier transform infrared imaging spectroscopy (FT-IRIS) can be used to assess the molecular composition of harvested repair tissue. Furthermore, use of an infrared fiber-optic probe (IFOP) has the potential for translation to a clinical setting to provide molecular information in situ. In the current study, we determined the feasibility of IFOP assessment of cartilage repair tissue in a rabbit model, and assessed correlations with gold-standard histology. Design. Bilateral osteochondral defects were generated in mature white New Zealand rabbits, and IFOP data obtained from defect and adjacent regions at 2, 4, 6, 8, 12, and 16 weeks postsurgery. Tissues were assessed histologically using the modified O’Driscoll score, by FT-IRIS, and by partial least squares (PLS) modeling of IFOP spectra. Results. The FT-IRIS parameters of collagen content, proteoglycan content, and collagen index correlated significantly with modified O’Driscoll score (P = 0.05, 0.002, and 0.02, respectively), indicative of their sensitivity to tissue healing. Repair tissue IFOP spectra were distinguished from normal tissue IFOP spectra in all samples by PLS analysis. However, the PLS model for prediction of histological score had a high prediction error, which was attributed to the spectral information being acquired from the tissue surface only. Conclusion. The strong correlations between FT-IRIS data and histological score support further development of the IFOP technique for clinical applications, although further studies to optimize data collection from the full sample depths are required

Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span

22 páginas, 4 figuras.A small molecule that safely mimics the ability of dietary restriction (DR) to delay age-related diseases in laboratory animals is greatly sought after. We and others have shown that resveratrol mimics effects of DR in lower organisms. In mice, we find that resveratrol induces gene expression patterns in multiple tissues that parallel those induced by DR and every-other-day feeding. Moreover, resveratrol-fed elderly mice show a marked reduction in signs of aging, including reduced albuminuria, decreased inflammation, and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density. However, mice fed a standard diet did not live longer when treated with resveratrol beginning at 12 months of age. Our findings indicate that resveratrol treatment has a range of beneficial effects in mice but does not increase the longevity of ad libitum-fed animals when started midlife.This work was supported by grants from the American Heart Association (0425834T to J.A.B. and 0435140N to A.C.) and from the NIH (RO1GM068072, AG19972, and AG19719 to D.A.S.), (HL077256 to Z.U.), (HD034089 to L.W), (2RO1 EY011733 to N.S.W.), Spanish grant (BFU2005-03017 to P.N.), and by the generous support of Mr. Paul F. Glenn and The Paul F. Glenn Laboratories for the Biological Mechanisms of Aging.Peer reviewe

Applications of Vibrational Spectroscopy for Analysis of Connective Tissues

Advances in vibrational spectroscopy have propelled new insights into the molecular composition and structure of biological tissues. In this review, we discuss common modalities and techniques of vibrational spectroscopy, and present key examples to illustrate how they have been applied to enrich the assessment of connective tissues. In particular, we focus on applications of Fourier transform infrared (FTIR), near infrared (NIR) and Raman spectroscopy to assess cartilage and bone properties. We present strengths and limitations of each approach and discuss how the combination of spectrometers with microscopes (hyperspectral imaging) and fiber optic probes have greatly advanced their biomedical applications. We show how these modalities may be used to evaluate virtually any type of sample (ex vivo, in situ or in vivo) and how “spectral fingerprints” can be interpreted to quantify outcomes related to tissue composition and quality. We highlight the unparalleled advantage of vibrational spectroscopy as a label-free and often nondestructive approach to assess properties of the extracellular matrix (ECM) associated with normal, developing, aging, pathological and treated tissues. We believe this review will assist readers not only in better understanding applications of FTIR, NIR and Raman spectroscopy, but also in implementing these approaches for their own research projects

Early Alterations in Bone Characteristics of Type I Diabetic Rat Femur: A Fourier Transform Infrared (FT-IR) Imaging Study

Alterations in microstructure and mineral features can affect the mechanical and chemical properties of bones and their capacity to resist mechanical forces. Controversial results on diabetic bone mineral content have been reported and little is known about the structural alterations in collagen, maturation of apatite crystals, and carbonate content in diabetic bone. This current study is the first to report the mineral and organic properties of cortical, trabecular, and growth plate regions of diabetic rat femurs using Fourier transform infrared (FT-IR) microspectroscopy and the Vickers microhardness test. Femurs of type I diabetic rats were embedded into polymethylmethacrylate blocks, which were used for FT-IR imaging and microhardness studies. A lower mineral content and microhardness, a higher carbonate content especially labile type carbonate content, and an increase in size and maturation of hydroxyapatite crystals were observed in diabetic femurs, which indicate that diabetes has detrimental effects on bone just like osteoporosis. There was a decrease in the level of collagen maturity in diabetic femurs, implying a decrease in bone collagen quality that may contribute to the decrease in tensile strength and bone fragility. Taken together, the findings revealed alterations in structure and composition of mineral and matrix components, and an altered quality and mechanical strength of rat femurs in an early stage of type I diabetes. The results contribute to the knowledge of structure-function relationship of mineral and matrix components in diabetic bone disorder and can further be used for diagnostic or therapeutic purposes