Infrared attenuated total reflection spectroscopic surface analysis of bovine‐tail intervertebral discs after UV‐light‐activated riboflavin‐induced collagen cross‐linking

The tensile strength of the intervertebral disc (IVD) is mainly maintained by collagen cross‐links. Loss of collagen cross‐linking combined with other age‐related degenerative processes contributes to tissue weakening, biomechanical failure, disc herniation and pain. Exogenous collagen cross‐linking has been identified as an effective therapeutic approach for restoring IVD tensile strength. The current state‐of‐the‐art method to assess the extent of collagen cross‐linking in tissues requires destructive procedures and high‐performance liquid chromatography. In this study, we investigated the utility of infrared attenuated total reflection (IR‐ATR) spectroscopy as a nondestructive analytical strategy to rapidly evaluate the extent of UV‐light‐activated riboflavin (B2)‐induced collagen cross‐linking in bovine IVD samples. Thirty‐five fresh bovine‐tail IVD samples were equally divided into five treatment groups: (a) untreated, (b) cell culture medium Dulbecco's Modified Eagle's Medium only, (c) B2 only, (d) UV‐light only and (e) UV‐light‐B2. A total of 674 measurements have been acquired, and were analyzed via partial least squares discriminant analysis. This classification scheme unambiguously identified individual classes with a sensitivity >91% and specificity >92%. The obtained results demonstrate that IR‐ATR spectroscopy reliably differentiates between different treatment categories, and promises an excellent tool for potential in vivo, nondestructive and real‐time assessment of exogenous IVD cross‐linking.     

The structural analysis of three‐dimensional fibrous collagen hydrogels by raman microspectroscopy

To investigate molecular effects of 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC), EDC/N‐hydroxysuccinimide (NHS), glyceraldehyde cross‐linking as well as polymerization temperature and concentration on the three‐dimensional (3D) collagen hydrogels, we analyzed the structures in situ by Raman microspectroscopy. The increased intensity of the 814 and 936 cm^?1 Raman bands corresponding to the C—C stretch of a protein backbone and a shift in the amide III bands from 1241 cm^?1/1268 cm^?1 in controls to 1247 cm^?1/1283 cm^?1 in glyceraldehyde‐treated gels indicated changes to the alignment of the collagen molecules, fibrils/fibers and/or changes to the secondary structure on glyceraldehyde treatment. The increased intensity of 1450 cm^?1 band and the appearance of a strong peak at 1468 cm^?1 reflected a change in the motion of lysine/arginine CH₂ groups. For the EDC‐treated collagen hydrogels, the increased intensity of 823 cm^?1 peak corresponding to the C—C stretch of the protein backbone indicated that EDC also changed the packing of collagen molecules. The 23% decrease in the ratio of 1238 cm^?1 to 1271 cm^?1 amide III band intensities in the EDC‐modified samples compared with the controls indicated changes to the alignment of the collagen molecules/fibrils and/or the secondary structure. A change in the motion of lysine/arginine CH₂ groups was detected as well. The addition of NHS did not induce additional Raman shifts compared to the effect of EDC alone with the exception of a 1416 cm^?1 band corresponding to a COO^? stretch. Overall, the Raman spectra suggest that glyceraldehyde affects the collagen states within 3D hydrogels to a greater extent compared to EDC and the effects of temperature and concentration are minimal and/or not detectable. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 349–356, 2013.     

Rapid intra‐operative diagnosis of kidney cancer by attenuated total reflection infrared spectroscopy of tissue smears

Herein, a technique to analyze air‐dried kidney tissue impression smears by means of attenuated total reflection infrared (ATR‐IR) spectroscopy is presented. Spectral tumor markers—absorption bands of glycogen—are identified in the ATR‐IR spectra of the kidney tissue smear samples. Thin kidney tissue cryo‐sections currently used for IR spectroscopic analysis lack such spectral markers as the sample preparation causes irreversible molecular changes in the tissue. In particular, freeze‐thaw cycle results in degradation of the glycogen and reduction or complete dissolution of its content. Supervised spectral classification was applied to the recorded spectra of the smears and the test spectra were classified with a high accuracy of 92% for normal tissue and 94% for tumor tissue, respectively. For further development, we propose that combination of the method with optical fiber ATR probes could potentially be used for rapid real‐time intra‐operative tissue analysis without interfering with either the established protocols of pathological examination or the ordinary workflow of operating surgeon. Such approach could ensure easier transition of the method to clinical applications where it may complement the results of gold standard histopathology examination and aid in more precise resection of kidney tumors.      

Three-dimensional organization of the collagen fibrils in the rat sciatic nerve as revealed by transmission- and scanning electron microscopy

Summary The organization of collagen fibrils in the rat sciatic nerve was studied by scanning electron microscopy after digestion of cellular elements by sodium hydroxide treatment, and by conventional transmission electron microscopy. The epineurium consisted mainly of thick bundles of collagen fibrils measuring about 10–20 m in width; they were wavy and ran slightly obliquely to the nerve axis. Between these collagen bundles, a very coarse meshwork of randomly oriented collagen fibrils was present. In the perineurium, collagen fibrils occupied the interspaces between the concentrically arranged perineurial cells; in each interspace, they formed a sheet of characteristic lacework elaborately interwoven by thin (about 3 m or less in width) bundles of collagen fibrils. In the subperineurial region, there was a distinct sheet of densely woven collagen fibrils between the perineurium and underlying endoneurial fibroblasts. In the endoneurium, collagen fibrils surrounded individual nerve fibers in two layers as scaffolds: the inner layer was made up of a delicate meshwork of very fine collagen fibrils, and the outer one consisted of longitudinally oriented bundles of about 1–3 m in width. The collagen fibril arrangement described above may protect the nerve fibers against external forces.     

Effect of SDS on human hair: Study on the molecular structure and morphology

This paper presents a model study to understand the effect of surfactants on the physicochemical properties of human hair. FT‐IR ATR spectroscopy has been employed to understand the chemical changes induced by sodium dodecyl sulfate (SDS) on human scalp hair. In particular, the SDS induced changes in the secondary structure of protein present in the outer protective layer of hair, i.e. cuticle, have been investigated. Conformational changes in the secondary structure of protein were studied by curve fitting of the amide I band after every phase of SDS treatment. It has been found that SDS brings rearrangements in the protein backbone conformations by transforming β ‐sheet structure to random coil and β ‐turn. Additionally, AFM and SEM studies were carried out to understand the morphological changes induced on the hair surface. SEM and AFM images demonstrated the rupture and partial erosion of cuticle sublayers. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Three-dimensional organization of fibroblasts and collagen fibrils in rat tail tendon

Summary The orderly arrangement of fibroblasts and collagen in tendons and ligaments suggests that these cells may have precise relationships with one another and with the collagen fibrils. The spatial organization of rat tail tendon was therefore examined using scanning and transmission electron microscopy and by reconstructing a 35-m long segment of tendon from serial transmission electron micrographs. Fibroblasts were regularly arranged in columns and showed more intimate association in the longitudinal than in the transverse plane. Thin cytoplasmic sheets extended up to 3 m transversely, frequently forming junctional attachments with similar processes from adjacent cells or from the same cell. Longitudinal processes were longer, often extending for more than 20 m and forming junctional attachments with other cells in the same column. Such processes often exhibited invaginations in which there were single fibrils or small groups of fibrils; this arrangement may be indicative of fibril elongation or may serve to transmit tension between the fibroblast and the collagen fibrils. This organization has interesting implications for the growth and function of other fibrous connective tissue, such as the periodontal ligament.     

Inside Cover: Surface analysis of sheep menisci after meniscectomy via infrared attenuated total reflection spectroscopy (J. Biophotonics 8/2019)

Infrared attenuated total reflection spectroscopy is an emerging label‐free method for analyzing the degree of damage in cartilage samples. In the present study, sheep menisci have been characterized after meniscectomy via the variances of relevant biomolecules at the incision surface. Further details can be found in the article by Angela I. López‐Lorente et al. ( e201800429 ).

     

Proteoglycan-type I collagen fibril interactions in bone and non-calcifying connective tissues

The association of proteogtycans with type I collagen fibrils in skin, tendon, cornea and bone has been determined by electron microscopy using an electrondense dye, Cupromeronic blue, in the critical electrolyte concentration mode, backed up by biochemical analysis and digestion by hyaluronidase or keratanase. A major proteoglycan of thesoft tissues, containing dermatan sulphat, is shown to be regularly and orthogonally arranged at the surface of the fibrils. Uranyl acetate counterstaining revealed that the main specific binding site is the d band, which previous work indicated is very close to the initial site of calcification of type I collagen fibrils. Bone, deminer-alized by a non-aqueous technique which preserves the proteoglycan in the tissue does not contain orthogonal arrays; the interfibrillar proteoglycan filaments are oriented parallel to the fibril axis. The main proteoglycan in bone is chondroitin sulphate-rich. It is suggested that dermatan sulphate proteoglycan plays a role in preventing soft connective tissues from calcifying.     

Minimally invasive screening for colitis using attenuated total internal reflectance fourier transform infrared spectroscopy

This article describes a rapid, simple and cost‐effective technique that could lead to a screening method for colitis without the need for biopsies or in vivo measurements. This screening technique includes the testing of serum using Attenuated Total Reflectance Fourier Transform Infrared (ATR‐FTIR) spectroscopy for the colitis‐induced increased presence of mannose. Chronic (Interleukin 10 knockout) and acute (Dextran Sodium Sulphate‐induced) models for colitis are tested using the ATR‐FTIR technique. Arthritis (Collagen Antibody Induced Arthritis) and metabolic syndrome (Toll like receptor 5 knockout) models are also tested as controls. The marker identified as mannose uniquely screens and distinguishes the colitic from the non‐colitic samples and the controls. The reference or the baseline spectrum could be the pooled and averaged spectra of non‐colitic samples or the subject's previous sample spectrum. This shows the potential of having individualized route maps of disease status, leading to personalized diagnosis and drug management.

     

Reconstituted type V collagen fibrils as cementing materials in the formation of cell clumps in culture

Previous studies have reported that type V collagen is an anti-adhesive substrate for cultured cells in that the cells detach from culture dishes coated with type V collagen molecules or polypeptides derived from them. We have noticed that human fetal lung fibroblasts (TIG-1) initially show no reduction in adherence to and spreading on a dish coated with reconstituted type V collagen fibrils but eventually detach from the dish and form cell clumps. To determine the way in which reconstituted type V collagen fibrils are involved in cell clump formation, we have followed the fate of the fluorescence of type V collagen fibrils pre-labeled with fluorescein isothiocyanate. Essentially, all the fluorescence disappeared from the dish surface as the cells detached and was condensed in the cell clumps. The cells that were recovered from clumps and dissociated into separate cells by trypsin treatment proliferated normally after they were seeded on a bare culture dish. This result and those from gel electrophoresis, fluorescence microscopy, and a cell proliferation assay indicate that the cell detachment from the dish is not caused by cell necrosis or apoptosis but by cellular motility together with the unique features of type V collagen fibrils. Not only the adherence of type V collagen fibrils to TIG-1 cells is much stronger than that to the culture dish, but the fibrils are retained on the cellular surface. The strong adherence of type V collagen fibrils to cells plays a role in cementing TIG-1 cells together.The present study was supported in part by Grant-in-Aid for Developmental Scientific Research (07558249), by The Japan Society for the Promotion of Science, Research for the Future Program (JSPS-RFTF96I00201), by the Program for Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR), by Grant-in-Aid for the Creation of Innovations through Business-Academic-Public Sector Cooperation to T.H., and by Grant-in-Aid for Scientific Research (B) to Y.I.     

Sum frequency vibrational spectroscopy: the molecular origins of the optical second-order nonlinearity of collagen

The molecular origins of second-order nonlinear effects in type I collagen fibrils have been identified with sum-frequency generation vibrational spectroscopy. The dominant contributing molecular groups are: 1), the methylene groups associated with a Fermi resonance between the fundamental symmetric stretch and the bending overtone of methylene; and 2), the carbonyl and peptide groups associated with the amide I band. The noncentrosymmetrically aligned methylene groups are characterized by a distinctive tilt relative to the axis perpendicular to the main axis of the collagen fiber, a conformation producing a strong achiral contribution to the second-order nonlinear effect. In contrast, the stretching vibration of the carbonyl groups associated with the amide I band results in a strong chiral contribution to the optical second-order nonlinear effect. The length scale of these chiral effects ranges from the molecular to the supramolecular.     

Collagen and proteoglycan in a sea urchin ligament with mutable mechanical properties

Summary The problematic ligament of sea urchins is a connective tissue which crosses the ball-and-socket joint between spine and body wall. The problem of this ligament is that it is composed of parallel collagen fibrils, yet normally undergoes rapid and dramatic alterations in mechanical properties and in length. Previous work has suggested that the collagen fibrils of the ligament are able to slide past one another during length changes but are inhibited from sliding when the ligament is in catch. In this model of the ligament both the collagen fibrils and the interfibrillar matrix are mechanically important. We have found that the collagen fibrils of the spine ligament of the pencil urchin Eucidaris tribuloides are discontinuous and end by tapering within the body of the ligament. Intact fibrils that have been isolated from the ligament vary by more than an order of magnitude in length and in radius but have a constant length/radius (aspect) ratio of about 5300. This is the first determination of the aspect ratio of collagen fibrils from any source. The constant aspect ratio of the fibrils is consistent with their functioning as the discontinuous fiber phase in a fiber-reinforced composite material, while the high value of the aspect ratio indicates that the nonfibrillar matrix, which must act to transfer stress between fibrils, can produce a stiff and strong ligament even if it is several orders of magnitude weaker and more compliant than the fibrils. Moreover, the tensile properties of the ligament may be determined by the properties of the matrix. A prominent component of the interfibrillar matrix is a proteoglycan which associates with specific bands at the surface of the collagen fibrils through noncovalent binding of its core protein. The glycosaminoglycan moiety of this proteoglycan is partly comprised of chondroitin sulfate/dermatan sulfate polymers. These results are consistent with the sliding fibril hypothesis and suggest that the proteoglycan may be an important component of the stress-transfer matrix.     

Effect of Mid-infrared Free-Electron Laser Irradiation on Refolding of Amyloid-Like Fibrils of Lysozyme into Native Form

Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610?C1,630?cm^?1, characteristic of a ??-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a ??-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620?cm^?1 (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70?C90?% of the native form. Both irradiations at 1,535?cm^?1(amide II band) and 1,240?cm^?1 (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000?cm^?1 afforded only about 60?% recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.     

Quantitative IR microscopy and spectromics open the way to 3D digital pathology

Currently, only mass‐spectrometry (MS) microscopy brings a quantitative analysis of chemical contents of tissue samples in 3D. Here, the reconstruction of a 3D quantitative chemical images of a biological tissue by FTIR spectro‐microscopy is reported. An automated curve‐fitting method is developed to extract all intense absorption bands constituting IR spectra. This innovation benefits from three critical features: (1) the correction of raw IR spectra to make them quantitatively comparable; (2) the automated and iterative data treatment allowing to transfer the IR‐absorption spectrum into a IR‐band spectrum; (3) the reconstruction of an 3D IR‐band matrix (x, y, z for voxel position and a 4^th dimension with all IR‐band parameters). Spectromics, which is a new method for exploiting spectral data for tissue metadata reconstruction, is proposed to further translate the related chemical information in 3D, as biochemical and anatomical tissue parameters. An example is given with oxidative stress distribution and the reconstruction of blood vessels in tissues. The requirements of IR microscopy instrumentation to propose 3D digital histology as a clinical routine technology is briefly discussed.

     

Rapid formation of myometrial gap junctions during parturition in the unilaterally implanted rat uterus

Summary In uterine smooth muscles, gap junction plaques rapidly form during the final stages of gestation. To investigate the related mechanisms, regional differences in myometrial gap junction development in rat uterus were examined quantitatively during delivery, using thin-section and freeze-fracture techniques in combination with light- and electron microscopy.Examination of implanted and nonimplanted horns in the unilaterally ligated rat bicornuate uteri, revealed no differences in the occurrence of gap junction plaques, but after 2 to 4 pups had been delivered, the contracted segments contained more gap junction plaques than did noncontracted segments examined immediately before delivery. In all segments, gap junctions were found more frequently in the circular muscle layers than in the longitudinal muscle layers. Gap junctions ranged in size from 0.002 m² to 0.52 m², but two-thirds were less than 0.1 m². The frequency of small gap junction plaques (less than 0.1 m²) was higher in the noncontracted segment.These results suggest that gap junctions are dynamic structures, and that their formation is controlled not only by general hormonal factors, possibly involved in gap junction increases in the myometrium before delivery, but also by local factors, possibly related to the contraction, that may accelerate an increase in gap junction formation during delivery.     

Fourier transform IR spectroscopy of collagen and gelatin solutions: deconvolution of the amide I band for conformational studies

The ir spectra of lathyritic rat skin collagen and calf skin gelatin solutions at a variety of temperatures were obtained using Fourier transform ir spectroscopy and a 9-reflection, 2-pass ZnSe prism sample cell. The spectra were then deconvolved (based on Kauppinnen's method) and the behavior of the amide I band at ～ 1650 cm^?1 observed in detail. Throughout the temperature range studied (4–50°C), three component absorption peaks within the amide I band (at 1633, 1643, and 1660 cm^?1) are common to the spectra irrespective of the degree of triple helix content of the sample. Changes in the relative intensities of these component peaks are, however, conformationally dependent. During denaturation of the triple helix, the dominant 1660-cm^?1 component in the native collagen spectrum diminishes and the 1633-cm^?1 peak becomes relatively intensified. The inherently strong basicity of the carbonyl group of the proline residues together with the frequent occurrence of this imino acid in the X position of the Gly-X-Y triplet of collagen largely accounts for the ?30-cm^?1 shift of the amide I band during denaturation. Temperature and conformationally dependent changes in the fine structure of the amide I band from dilute solutions of collagen can be monitored in a reproducible and quantitative fashion.     

Near‐infrared spectroscopic evaluation of lyophilized viral vaccine formulations

This article examines the applicability of near‐infrared spectroscopy (NIRS) to evaluate the virus state in a freeze‐dried live, attenuated vaccine formulation. Therefore, this formulation was freeze‐dried using different virus volumes and after applying different pre‐freeze‐drying virus treatments (resulting in different virus states): (i) as used in the commercial formulation; (ii) without antigen (placebo); (iii) concentrated via a centrifugal filter device; and (iv) stressed by 96 h exposure to room temperature. Each freeze‐dried product was measured directly after freeze‐drying with NIR spectroscopy and the spectra were analyzed using principal component analysis (PCA). Herewith, two NIR spectral regions were evaluated: (i) the 7300–4000 cm^?1 region containing the amide A/II band which might reflect information on the coated proteins of freeze‐dried live, attenuated viruses; and (ii) the C–H vibration overtone regions (10,000–7500 and 6340–5500 cm^?1) which might supply information on the lipid layer surrounding the freeze‐dried live, attenuated viruses. The different pre‐freeze‐drying treated live, attenuated virus formulations (different virus states and virus volumes) resulted in different clusters in the scores plots resulting from the PCA of the collected NIR spectra. Secondly, partial least squares discriminant analysis models (PLS‐DA) were developed and evaluated, allowing classification of the freeze‐dried formulations according to virus pretreatment. The results of this study suggest the applicability of NIR spectroscopy for evaluating live, attenuated vaccine formulations with respect to their virus pretreatment and virus volume. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1573–1586, 2013     

Hemodialysis monitoring using mid‐ and near‐infrared spectroscopy with partial least squares regression

Blood constituents such as urea, glucose, lactate, phosphate and creatinine are of high relevance in monitoring the process of detoxification in ambulant dialysis treatment. In the present work, 2 different vibrational spectroscopic techniques are used to determine those molecules quantitatively in artificial dialysate solutions. The goal of the study is to compare the performance of near‐infrared (NIR) and mid‐infrared (MIR) spectroscopy in hyphenation with partial least squares regression (PLSR) directly by using the same sample set. The results show that MIR spectroscopy is better suited to analyze the analytes of interest. Multilevel multifactor design is used to cover the relevant concentration variations during dialysis. MIR spectroscopy coupled to a multi reflection attenuated total reflection (ATR) cell enables reliable prediction of all target analytes. In contrast, the NIR spectroscopic method does not give access to all 5 components but only to urea and glucose. For both methods, coefficients of determination greater or equal to 0.86 can be achieved in the test‐set validation process for urea and glucose. Lactate, phosphate and creatinine perform well in the MIR with R² ≥ 0.95 using test‐set validation.      

Noninvasive assessments of skin glycated proteins by fluorescence and Raman techniques in diabetics and nondiabetics

Diabetes is a complex metabolic disease and has chronic complications. It has been considered a serious public health problem. The aim of the current study was to evaluate skin glycated proteins through fluorescence and Raman techniques. One hundred subjects were invited to participate in the study. Six volunteers did not attend due to exclusion criteria or a change of mind about participating. Therefore, 94 volunteers were grouped according to age range (20‐80 years), health condition (nondiabetic, with insulin resistance [IR] and/or diabetic) and Fitzpatrick skin type (I‐VI). The fluorescence spectrometer and the portable Raman spectroscopy system were used to measure glycated proteins from the skin. There was elevated skin autofluorescence in healthy middle‐aged and elderly subjects, as well as in patients with IR and/or diabetes. Regarding Raman spectroscopy, changes in the skin hydration state, degradation of type I collagen and greater glycation were related for diabetes and chronological aging. Weak and positive correlation between the skin autofluorescence and the Raman peaks ratio (855/876) related to the glycated proteins was also found. Raman spectroscopy shows several bands for spectral analyses, complementing the fluorescence data. Therefore, this study contributes to understanding of the optical of human skin for noninvasive diabetes screening.      

Experimental and computational studies of enantioseparation of structurally similar chiral compounds on amylose tris(3,5‐dimethylphenylcarbamate)

The enantioseparation of 14 structurally similar chiral solutes, with one or two chiral centers, are studied for a commercially important polysaccharide‐based chiral stationary phase, amylose tris(3,5‐dimethylphenylcarbamate) (ADMPC). Among these solutes, only two solutes show significant enantioresolutions of 2 to 2.5 in n‐hexane/2‐propanol (90/10, v/v) at 298 K. The retention factors of the chiral solutes vary significantly from 0.7 to 7.0, and they are compared with those of simpler nonchiral solutes having similar but fewer functional groups. The sorbent–solute H‐bonding interactions between the solute functional groups and the polymer C?O and NH functional groups are probed with attenuated total reflection infrared spectroscopy (ATR‐IR). The H‐bonding interactions of the polymer C?O and NH groups with the solutes result in changes in the IR amide band wavenumbers of ADMPC upon solute adsorption. The nanostructure of an ADMPC cavity and the potential interactions with the chiral solutes are proposed based on the sorbent–solute–solvent HPLC data, the sorbent–solute IR data, and the sorbent–solute molecular dynamics (MD) simulations. The results are consistent with the three point attachment hypothesis and indicate that a significant enantioresolution in ADMPC requires at least three different interaction sites for simultaneous H‐bonds and phenyl–phenyl interactions for phenylpropylamine (PPA) and various structurally similar chiral solutes. Chirality 2010. © 2009 Wiley‐Liss, Inc.