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Quantitative Raman characterization of cross-linked collagen thin films as a model system for diagnosing early osteoarthritis
Abstract
The onset of osteoarthritis (OA)in articular cartilage is characterized by degradation of extracellular matrix (ECM). Specifically, breakage of cross-links between collagen fibrils in the articular cartilage leads to loss of structural integrity of the bulk tissue. Since there are no broadly accepted, non-invasive, label-free tools for diagnosing OA at its early stage, Raman spectroscopyis therefore proposed in this work as a novel, non-destructive diagnostic tool. In this study, collagen thin films were employed to act as a simplified model system of the cartilage collagen extracellular matrix. Cross-link formation was controlled via exposure to glutaraldehyde (GA), by varying exposure time and concentration levels, and Raman spectral information was collected to quantitatively characterize the cross-link assignments imparted to the collagen thin films during treatment. A novel, quantitative method was developed to analyze the Raman signal obtained from collagen thin films. Segments of Raman signal were decomposed and modeled as the sum of individual bands, providing an optimization function for subsequent curve fitting against experimental findings. Relative changes in the concentration of the GA-induced pyridinium cross-links were extracted from the model, as a function of the exposure to GA. Spatially resolved characterization enabled construction of spectral maps of the collagen thin films, which provided detailed information about the variation of cross-link formation at various locations on the specimen. Results showed that Raman spectral data correlate with glutaraldehyde treatment and therefore may be used as a proxy by which to measure loss of collagen cross-links in vivo. This study proposes a promising system of identifying onset of OA and may enable early intervention treatments that may serve to slow or prevent osteoarthritis progression.
... Prevalent interventions for corneal ectasia include corneal collagen crosslinking and intraocular contact lens implantation (IOL). The former is of particular interest as the mechanical properties and structural stability of collagen rich tissues, such as cornea and articular cartilage [14][15][16], are significantly influenced by the collagen cross-linking [17]. ...
A new paradigm for strengthening of corneal tissue as well as permanent correction of refractive errors has been proposed. Ultrafast laser irradiation is confined to the levels below optical breakdown such that tissue damage is avoided while creating an ionization field responsible for subsequent photochemical modification of the stroma. The concept was assed using newly developed platform for precise application of a near-IR femtosecond laser irradiation to the cornea in in-vitro experiments. Targeted irradiation with tightly focused ultrafast laser pulses allows spatially resolved crosslinking in the interior of the porcine cornea in the absence of photosensitizers. The formation of intra- or interstromal covalent bonds in collagen matrix locally increases lamellar density. Due to high resolution, treatment is spatially resolved and therefore can be tailored to either enhance structure of corneal stroma or adjust corneal curvature towards correcting refractive errors. As the induced modification is primarily driven by nonlinear absorption, the treatment is essentially wavelength independent, and as such potentially less harmful than current method of choice, joint application of UVA light irradiation in conjunction with riboflavin. Potential applicability of a near-IR femtosecond laser for biomechanical stabilization of cornea and non-invasive refractive eye corrections is discussed.
Vibrational spectroscopic methods are minimally invasive, and are appropriate for use in clinical contexts. Methods were developed in this dissertation for evaluating joint damage and disease using Raman spectroscopy. Subtle changes in the molecular structure of joint tissue and synovial fluid precede morphological changes in the joint. The goal of this research is to develop Raman spectroscopic methods for the examination of joint tissue and biological fluids, for monitoring and detecting molecular alterations associated with osteoarthritis. We identified Raman spectroscopic markers of altered molecular structure in subchondral bone and relevant biological fluids. Using Raman spectroscopy the molecular structure of joint tissues was measured, and the results were compared to the results from micro computed tomographic and histopathologic analysis. Raman spectra of subchondral bone collected from Del1 (+/-) transgenic mice, a mouse model for early-onset osteoarthritis, indicated lower bone mineralization in transgenic mice (5.73 ?? 0.28 vs. 6.87 ?? 0.225 in wild-type mice, p=0.003). A fiber-optic Raman probe for arthroscopic measurements was developed to demonstrate the feasibility of measuring the molecular structure of joint tissue with clinically-relevant instrumentation. The carbonate-to-phosphate ratio, a Raman spectroscopic measurement of bone mineral composition, was measured from subchondral bone under an intact layer of cartilage. Our initial work on a human proximal radius specimen indicated that an arthroscope configuration is capable of providing similar carbonate-to-phosphate values as spectra collected on a Raman microscope (0.25 vs. 0.24). In addition to cartilage and subchondral bone, the chemical structure of synovial fluid molecules is a key factor in maintaining healthy joint function. Synovial fluid from normal and diseased joints was examined using a novel drop deposition/Raman spectroscopic method. Raman spectra of synovial fluid from patients with radiographic evidence of osteoarthritis showed evidence of altered protein structure, as shown by increased Raman band intensity ratios at 1080 cm-1/1002 cm-1 (0.054 ?? 0.07 vs. 0.038 ?? 0.003, p
Many current algorithms for - nonlinear constrained optimization problems determine a direction by solving a quadratic programming subproblem. The global convergence properties are addressed by using a line search technique and a merit function to modify the length of the step obtained from the quadratic program. In unconstrained optimization trust regions strategies have been very successful. In this paper we present a new approach for equality constrained optimization problems based on a trust region strategy. The direction selected is not necessarily the solution of the standard quadratic programming subproblem.
Type I and IV collagens are important constituents of the skin. Type I collagen is found in all dermal layers in high proportion, while type IV collagen is localized in the basement membrane of the dermo-epidermal junction (DEJ). These proteins are strongly altered during aging or cancer progression. Although they possess amino acid compositions which, are close, they present also important structural differences inducing specific physicochemical properties. Raman spectroscopy is based on a nondestructive interaction of the light with the matter. This technique permits to probe the intrinsic molecular composition of the samples without staining or particular preparation. The aim of our research is to study the correlation between the molecular conformations of type I and IV collagens and their Raman features. We showed that signals specific of each protein can be revealed and that they translate structural differences between the two collagens. From this collagens spectral characterization, the analysis of skin sections also permitted to identify spectral markers of dermis, epidermis, and epidermis/dermis interface. These preliminary results represent basic data for further studies, particularly to probe skin molecular alterations induced by chronologic aging.
Osteoarthritis is a prevalent and disabling disease affecting an increasingly large swathe of the world population. While clinical osteoarthritis is a late-stage condition for which disease-modifying opportunities are limited, osteoarthritis typically develops over decades, offering a long window of time to potentially alter its course. The etiology of osteoarthritis is multifactorial, showing strong associations with highly modifiable risk factors of mechanical overload, obesity and joint injury. As such, characterization of pre-osteoarthritic disease states will be critical to support a paradigm shift from palliation of late disease towards prevention, through early diagnosis and early treatment of joint injury and degeneration to reduce osteoarthritis risk. Joint trauma accelerates development of osteoarthritis from a known point in time. Human joint injury cohorts therefore provide a unique opportunity for evaluation of pre-osteoarthritic conditions and potential interventions from the earliest stages of degeneration. This review focuses on recent advances in imaging and biochemical biomarkers suitable for characterization of the pre-osteoarthritic joint as well as implications for development of effective early treatment strategies.
1-(5-Amino-5-carboxypentyl)pyridinium chloride (3) and 1-(5-amino-5-carboxypentyl)-3-[1-(5-amino-5-carboxypentyl)-2-piperidyl]pyridinium chloride, anabilysine (4), have been isolated from acid hydrolysates of glutaraldehyde-treated ovalbumin and their structures confirmed by synthesis. It is concluded that the strong chromophore at ca. 265 nm shown by glutaraldehyde-treated proteins is due to the formation of such quaternary pyridinium compounds and that anabilysine residues are important in the cross-linking of proteins by glutaraldehyde.
Publisher Summary
Most carbonyl compounds have infrared (IR) and Raman bands in the 1900–1550 cm−1 region. In the infrared, C=O bands tend to be among the strongest in the spectra whereas in the Raman, they are considerably weaker. When there are two C=O groups in a molecule with a center of symmetry, then the antisymmetric C=O stretch is only active in the IR and the symmetric stretch is only active in the Raman. The intensity of the C=O band in the IR makes the detection of the carbonyl group easier in the infrared spectrum particularly when the carbonyl group is present in small amounts. Raman intensities of C=O bands tend to be dependent mainly on resonance effects and are little affected by inductive effect. This chapter discusses the factors that shift carbonyl bands. The C=O stretching frequency is relatively insensitive to a change in the mass of an atom directly attached to the C=O carbon as long as that atom is carbon or a heavier atom.
Fluorescent background is a major problem in recoding the Raman spectra of many samples, which swamps or obscures the Raman signals. The background should be suppressed in order to perform further qualitative or quantitative analysis of the spectra. For this purpose, an intelligent background-correction algorithm is developed, which simulates manual background-correction procedure intelligently. It basically consists of three aspects: (1) accurate peak position detection in the Raman spectrum by continuous wavelet transform (CWT) with the Mexican Hat wavelet as the mother wavelet; (2) peak-width estimation by signal-to-noise ratio (SNR) enhancing derivative calculation based on CWT but with the Haar wavelet as the mother wavelet; and (3) background fitting using penalized least squares with binary masks. This algorithm does not require any preprocessing step for transforming the spectrum into the wavelet space and can suppress the fluorescent background of Raman spectra intelligently and validly. The algorithm is implemented in R language and available as open source software (). Copyright ?2009 John Wiley & Sons, Ltd.
This review has focused on the prevalence and risk factors associated with knee and hip osteoarthritis. Risk factors for knee osteoarthritis are obesity and major injury, and knee osteoarthritis probably fits into the generalized osteoarthritis diathesis. Repetitive use, such as in jobs requiring heavy labor and knee bending, probably increases the risk of knee osteoarthritis. Hip osteoarthritis is probably frequently secondary to developmental defects. As Rothman (182) has pointed out in discussing causation, this does not necessarily mean that the same factors do not also contribute to causing hip osteoarthritis. Yet, it appears that, in many cases, developmental defects are severe enough to be sufficient causes of hip osteoarthritis. To delineate other causes, it may be necessary to examine risk factors separately in those with and in those without developmental disease. Although large epidemiologic studies are best able to identify the relative contributions of specific risk factors while controlling for other risk factors, new studies need to focus on important unresolved questions. First, longitudinal studies with comprehensive follow-up using repeated radiographic assessments are needed to identify factors that cause development of disease or the onset of symptoms. Second, cohorts with early and possibly asymptomatic disease need to be followed to determine the causes of progression or regression of disease and the natural history of disease. Such cohorts may include those at high risk of injury such as sports enthusiasts or manual laborers.
We report the isolation and chemical characterization of collagen cross-linking compounds, 3-hydroxypyridinium and dihydroxylysinonorleucine, from human urine.
Type I collagen is the most abundant protein in humans, and it helps to maintain the integrity of many tissues via its interactions with cell surfaces, other extracellular matrix molecules, and growth and differentiation factors. Nearly 50 molecules have been found to interact with type I collagen, and for about half of them, binding sites on this collagen have been elucidated. In addition, over 300 mutations in type I collagen associated with human connective tissue disorders have been described. However, the spatial relationships between the known ligand-binding sites and mutation positions have not been examined. To this end, here we have created a map of type I collagen that includes all of its ligand-binding sites and mutations. The map reveals the existence of several hot spots for ligand interactions on type I collagen and that most of the binding sites locate to its C-terminal half. Moreover, on the collagen fibril some potentially relevant relationships between binding sites were observed including the following: fibronectin- and certain integrin-binding regions are near neighbors, which may mechanistically relate to fibronectin-dependent cell-collagen attachment; proteoglycan binding may potentially impact upon collagen fibrillogenesis, cell-collagen attachment, and collagen glycation seen in diabetes and aging; and mutations associated with osteogenesis imperfecta and other disorders show apparently nonrandom distribution patterns within both the monomer and fibril, implying that mutation positions correlate with disease phenotype. These and other observations presented here may provide novel insights into evaluating type I collagen functions and the relationships between its binding partners and mutations.
Raman spectroscopy is a vibrational spectroscopic technique that can be used to optically probe the molecular changes associated with diseased tissues. The objective of our study was to explore near-infrared (NIR) Raman spectroscopy for distinguishing tumor from normal bronchial tissue. Bronchial tissue specimens (12 normal, 10 squamous cell carcinoma (SCC) and 6 adenocarcinoma) were obtained from 10 patients with known or suspected malignancies of the lung. A rapid-acquisition dispersive-type NIR Raman spectroscopy system was used for tissue Raman studies at 785 nm excitation. High-quality Raman spectra in the 700-1,800 cm(-1) range from human bronchial tissues in vitro could be obtained within 5 sec. Raman spectra differed significantly between normal and malignant tumor tissue, with tumors showing higher percentage signals for nucleic acid, tryptophan and phenylalanine and lower percentage signals for phospholipids, proline and valine, compared to normal tissue. Raman spectral shape differences between normal and tumor tissue were also observed particularly in the spectral ranges of 1,000-1,100, 1,200-1,400 and 1,500-1,700 cm(-1), which contain signals related to protein and lipid conformations and nucleic acid's CH stretching modes. The ratio of Raman intensities at 1,445 to 1,655 cm(-1) provided good differentiation between normal and malignant bronchial tissue (p < 0.0001). The results of this exploratory study indicate that NIR Raman spectroscopy provides significant potential for the noninvasive diagnosis of lung cancers in vivo based on the optic evaluation of biomolecules.
Raman Spectrographic Characterization of Cartilage Matrix Swelling via Lysyl Oxidase Inhibition in Immature Explantsand Tissue Constructs. 2015.
- Durney
Quantitative analysis ofRaman spectra for assessment of crosslink concentrations toward diagnosing early osteoarthritis.
- Chao
Course notes on the interpretation of infrared and Raman spectra
- D W Mayo
- F A Miller
- R W Hannah
Synovial fluid andphysiologic levels of cortisol, insulin, and glucose in media maintain the homeostasis of immature bovine cartilage explants over long-term culture.
- Albro