The cross-linking and protective effect of artemisinin and its derivatives on collagen fibers of demineralized dentin surface.

OBJECTIVE: To investigate the cross-linking and protective effect of artemisinin (ART), dihydroartemisinin (DHA), and artesunate (AST) on collagen fibers of demineralized dentin surface. METHODS: Molecular docking was used to predict potential interactions of ART, DHA, and AST with dentin type I collagen. Human third molars without caries were completely demineralized and treated with different solutions for 1 min. The molecular interactions and cross-linking degree of ART and its derivatives with dentin collagen were evaluated by FTIR spectroscopy, total extractable protein content, and a ninhydrin assay. Scanning electron microscopy, hydroxyproline release, and ultimate microtensile strength tests (µUTS) were employed to confirm the mechanical properties and anti-collagenase degradation properties of dentin collagen fibers. RESULTS: ART, DHA, and AST combined with dentin type I collagen mainly through hydrogen bonding and hydrophobic interactions, and the cross-linking reaction sites were mainly C=O and CN functional groups. Compared to the control group, ART and its derivatives significantly increased the degree of cross-linking. Additionally, significant increases were observed in resistance to enzymatic digestion and mechanical properties of the artemisinin and its derivatives group. CONCLUSION: ART, DHA, and AST could cross-link with demineralized dentin collagen, through improving the mechanical properties and anti-collagenase degradation properties. CLINICAL SIGNIFICANCE: The study endorses the potential use of ART and its derivatives as a prospective collagen cross-linking agent for degradation-resistant and long-period dentin bonding in composite resin restorations.

Tracking chromatin state changes using nanoscale photo-proximity labelling.

Interactions between biomolecules underlie all cellular processes and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects1,2. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health1. However, in the complex environment of the nucleus, it is challenging to determine protein-protein interactions owing to low abundance, transient or multivalent binding and a lack of technologies that are able to interrogate these interactions without disrupting the protein-binding surface under study3. Here, we describe a method for the traceless incorporation of iridium-photosensitizers into the nuclear micro-environment using engineered split inteins. These Ir-catalysts can activate diazirine warheads through Dexter energy transfer to form reactive carbenes within an approximately 10 nm radius, cross-linking with proteins in the immediate micro-environment (a process termed µMap) for analysis using quantitative chemoproteomics4. We show that this nanoscale proximity-labelling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. µMap improves our fundamental understanding of nuclear protein-protein interactions and, in doing so, is expected to have a significant effect on the field of epigenetic drug discovery in both academia and industry.

Influence of two natural cross-linkers on microtensile bond strength durability ­ An in vitro study / Influência de dois reticuladores naturais na durabilidade da resistência de união à microtração ­ Um estudo in vitro

Objective: to investigate the effect of two natural cross-linkers on microtensile bond strength (µTBS) and evaluate their influence on the durability of the resin dentin bonds. Material and Methods: the Moringa oleifera and Centella asiatica plant extracts were qualitatively tested with high-performance thin layer chromatography (HPTLC) for the presence of phenols. The phenolic content ranged from 27 to 30 gallic acid equivalents (GAE), µg/mg of dry weight. After etching, two concentrations (5% and 1%) of these two extracts were prepared and used as pretreatment liners on dentin. They were applied for a min. After restoration with resin composite, dentin resin beams were prepared. The study groups were 5% Moringa, 1% Moringa 5% Centella 1% Centella, and control (without cross-linker application). For each group, half of the samples underwent µTBS testing after 24 hours, while the remaining half were immersed in artificial saliva to assess the bond's longevity after 6 months of ageing. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. Results: both 5% and 1% Moringa showed a significant difference (p<0.05) compared to the other groups at both intervals. However, after ageing, the specimens in the control and 1% Centella groups resulted in a significant decrease in µTBS. Conclusion: overall, both concentrations of Moringa (5% and 1%) were effective in stabilising the bond during both intervals.(AU)

Objetivo: investigar o efeito de dois reticuladores naturais na resistência de união (µTBS) à microtração e avaliar sua influência na durabilidade da adesão da resina à dentina. Material e Métodos: extratos das plantas Moringa oleifera e Centella asiatica foram qualitativamente testados através de cromatografia em camada fina de alta performance (HPTLC) para a presença de fenóis. O conteúdo fenólico alcançou entre 27 a 30 equivalentes de ácido gálico (GAE), µg/mg de peso seco. Após o condicionamento, duas concentrações (5% e 1%) dos extratos foram preparadas e utilizadas como forros de pré-tratamento em dentina. Eles foram aplicados por um minuto. Após a restauração com resina composta, palitos de dentina e resina foram preparados. Os grupos foram 5% Moringa, 1% Moringa, 5% Centella, 1% Centella e controle (sem aplicação de reticulador). Para cada grupo, metade das amostras foram submetidas ao teste µTBS após 24 horas, enquanto a outra metade foi imersa em saliva artificial para avaliar a longevidade adesiva após 6 meses de envelhecimento. Foi realizada análise estatística através de ANOVA 1-fator, seguido do teste post hoc de Tukey. Resultados: ambas as concentrações de 5% e 1% de Moringa demonstraram diferença significativa (p<0.05) comparadas aos outros grupos em ambos os intervalos. No entanto, após o envelhecimento, os espécimes dos geupos controle e 1% de Centella resultaram em uma redução significativa de µTBS. Conclusão: no geral, ambas as concentrações de Moringa (5% e 1%) foram efetivas em estabelecer a adesão em ambos os intervalos (AU)

Cross-linked polydimethylsiloxane colloid as novel contrast agent for gastrointestinal magnetic resonance imaging: Transient nuclear Overhauser effect within the interface.

With good contrast in T1 and T2 weighted imaging as well as low toxicity in 3- (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, this work proposes the cross-linked polydimethylsiloxane colloids as a novel non-ionic contrast agent for gastrointestinal magnetic resonance imaging. The experiments of nuclear magnetic resonance spectra and relaxation show that within the interface of the colloids, there are nuclear Overhauser effect and transient nuclear Overhauser effect (cross-relaxation). Regarding the longitudinal relaxation experiments of CH2CH2O segments of Tween 80, a two spins system is found and modeled well by the equation IZ-I0= S0((1-X) e-tD1 -(1+X) e-tT1) which is deduced based on the transient nuclear Overhauser effect proposed by Solomon. The arbitrary constant X is additionally added with the initial conditions (Iz - I0)t=0 = -2XS0 and (Sz - S0)t=0 = -2S0. For the two spins system, D1 and T1 are corresponding to longitudinal relaxation times of the bound water and the CH2CH2O respectively. Concerning the transverse relaxation experiments of the CH2CH2O, they agree with the equation with three exponential decays, defined by three relaxation times, likely corresponding to three mechanisms. These mechanisms possibly are intramolecular and intermolecular dipole-dipole (DD) interactions and scalar coupling. Within the interface, hydrogen bonding causes the positive nuclear Overhauser effect of the CH2CH2O's nuclear magnetic resonance spectra, the transient nuclear Overhauser effect of the CH2CH2O's longitudinal relaxation experiments and the intermolecular dipole-dipole interactions of the CH2CH2O's transverse relaxation experiments.

Identifying transglutaminase reaction products via mass spectrometry as exemplified by the MUC2 mucin - Pitfalls and traps.

In order to demonstrate transglutaminase activity in biological samples immunological as well as glutamine- and amine-donor based assays are commonly used. However, the identification of the transglutaminase reaction product, i. e. the isopeptide cross-linked peptides/proteins or the deamidated protein/peptide are often neglected. This article describes a workflow for the detection of the products of transglutaminase-catalyzed reactions. In particular, possible pitfalls and traps that can arise during the mass spectrometry-based identification of isopeptide cross-links are addressed and characterised on actual samples.

Determination of Triglycidyl Isocyanurate in Workplace Air.

Triglycidyl isocyanurate (TGIC) is a white solid in powder or granular form. TGIC does not occur naturally in the environment. It is intentionally manufactured and used as a crosslinking agent or hardener to produce polyester powder coatings. TGIC may cause genetic defects. This article presents the method of TGIC determination in workplace air using high-performance liquid chromatography (HPLC) with a diode-array detector (DAD). The method is based on the collection of TGIC present in the air on a polypropylene filter, extraction with acetonitrile, and chromatographic analysis of the solution obtained in this way. The determination was carried out in the reverse-phase system (mobile phase: acetonitrile: water) using an Ultra C18 column. The measurement range is 2 to 40 µg/m3 for a 720 liters air sample. Limit of detection (LOD) is 23 ng/m3 and limit of quantification (LOQ): 70 ng/m3. The method can be used for assessing occupational exposure to TGIC and associated risk to workers' health.

An untargeted evaluation of the volatile and semi-volatile compounds migrating into food simulants from polypropylene food containers by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.

This study reports on the potential of comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC×GC-ToF MS) for the exhaustive untargeted characterization of the volatile and semi-volatile analytes migrating from four commercial polypropylene food containers into four simulants (water, 3% acetic acid, 10% ethanol, and isooctane) according to European Regulation 10/2011. Collected extracts were concentrated and directly subjected to GC×GC-ToF MS analysis without any further treatment to preserve migrants integrity. As expected, the nature and total number of compounds detected in the migrates depended on both the brand (i.e., manufacture and/or sterilization procedure) and the simulant applied. In total, 107 analytes, including some less volatile compounds, were either positively or tentatively identified in the investigated simulants, a number of these compounds being reported for the first time as migrants from this type of material. A database containing chromatographic, mass spectral and partition information concerning these compounds, plus 23 remaining unidentified, is provided.

Correlation between Glyoxal-Induced DNA Cross-Links and Hemoglobin Modifications in Human Blood Measured by Mass Spectrometry.

Glyoxal is an oxoaldehyde generated from the degradation of glucose-protein conjugates and from lipid peroxidation in foods and in vivo, and it is also present in the environment (e.g., cigarette smoke). The major endogenous source of glyoxal is glucose autoxidation, and the glyoxal concentrations in plasma are higher in diabetic patients than in nondiabetics. Glyoxal reacts with biomolecules forming covalently modified DNA and protein adducts. We previously developed sensitive and specific assays based on nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) for quantification of DNA cross-linked adducts (dG-gx-dC and dG-gx-dA) and for hemoglobin adducts derived from glyoxal. In this study, we isolated and analyzed both leukocyte DNA and hemoglobin from the blood of diabetic patients and compared the adduct levels with those from nondiabetic subjects using the modified assays. The results indicated that the extents of glyoxal-induced hemoglobin modifications on α-Lys-11, α-Arg-92, ß-Lys-17, and ß-Lys-66 were statistically higher in diabetic patients than nondiabetics and they correlated with HbA1c significantly. Moreover, the levels of dG-gx-dC in leukocyte DNA correlated positively with the extents of globin modification at α-Lys-11 and ß-Lys-17, while levels of dG-gx-dA correlated with those at α-Lys-11 and α-Arg-92 in nonsmoking subjects. Comparing the levels and the correlation coefficients of these hemoglobin and DNA adducts including or excluding smokers, it appears that smoking is not a major contributor to glyoxal-induced adduction of hemoglobin and leukocyte DNA. To the best of our knowledge, this is one of the few reports of positive correlation between DNA and protein adducts of the same compound (glyoxal) in the blood from the same subjects. Because of the high abundance of hemoglobin in blood, the results indicate that quantification of glyoxal-modified peptides in hemoglobin might serve as a dosimetry for glyoxal and a practical surrogate biomarker for assessing glyoxal-induced DNA damage and its prevention.

Application of non-canonical crosslinking amino acids to study protein-protein interactions in live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) equipped with photo-crosslinking and chemical crosslinking moieties has found broad application in the study of protein-protein interactions from a unique perspective in live cells. We highlight here applications of photo-activatable ncAAs to map protein interaction surfaces and to capture protein-protein interactions, and we describe recent efforts to efficiently couple photo-crosslinking with mass spectrometric analysis. In addition, we describe recent advances in the development and application of ncAAs for chemical crosslinking, including protein stapling, photo-control of protein conformation, two-dimensional crosslinking, and stabilization of transient and low-affinity protein-protein interactions. We expect that the field will keep growing in the near future and enable the tackling of ambitious biological questions.

A high crosslinking grade of hyaluronic acid found in a dermal filler causing adverse effects.

Facial treatments with dermal fillers for medical or esthetic purposes occasionally give rise to adverse effects, ranging from temporary effects such as reddening of the skin, to long term effects such as hardening of tissue. There appears to be a relationship between the lifetime of the filler product and the risk for adverse effects. The lifetime of hyaluronic acid-based fillers is dependent on the presence and amount of crosslinking agents such as 1,4-butanediol diglycidyl ether (BDDE). It would therefore make sense to establish methodology to analyze the crosslinking grade of HA-based filler products on a routine basis. To this end, an analytical method was developed and validated to identify HA-BDDE-based fillers and to quantify their modification and crosslinking grade. The method was subsequently applied to products from the legal supply chain and the illegal market. It was found that the product Hyacorp H 1000, previously taken from the market, indeed contains a high modification grade and crosslinking grade, as was the assumed reason for the increased risk for adverse effects of this product. However, it was also shown that the Hyacorp products are highly unreliable in relation to their product composition in general. In this study, authentic products could not be distinguished from the illegal market products based on their modification and crosslinking grade.

Mango: A General Tool for Collision Induced Dissociation-Cleavable Cross-Linked Peptide Identification.

Chemical cross-linking combined with mass spectrometry provides a method to study protein structures and interactions. The introduction of cleavable bonds in a cross-linker provides an avenue to decouple released peptide masses from their precursor species, greatly simplifying the downstream search, allowing for whole proteome investigations to be performed. Typically, these experiments have been challenging to carry out, often utilizing nonstandard methods to fully identify cross-linked peptides. Mango is an open source software tool that extracts precursor masses from chimeric spectra generated using cleavable cross-linkers, greatly simplifying the downstream search. As it is designed to work with chimeric spectra, Mango can be used on traditional high-resolution tandem mass spectrometry (MS/MS) capable mass spectrometers without the need for additional modifications. When paired with a traditional proteomics search engine, Mango can be used to identify several thousand cross-linked peptide pairs searching against the entire Escherichia coli proteome. Mango provides an avenue to perform whole proteome cross-linking experiments without specialized instrumentation or access to nonstandard methods.

Characterization and Identification of Dityrosine Cross-Linked Peptides Using Tandem Mass Spectrometry.

The use of mass spectrometry coupled with chemical cross-linking of proteins has become a powerful tool for proteins structure and interactions studies. Unlike structural analysis of proteins using chemical reagents specific for lysine or cysteine residues, identification of gas-phase fragmentation patterns of endogenous dityrosine cross-linked peptides have not been investigated. Dityrosine cross-linking in proteins and peptides are clinical markers of oxidative stress, aging, and neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. In this study, we investigated and characterized the fragmentation pattern of a synthetically prepared dityrosine cross-linked dimer of Aß(1-16) using ESI tandem mass spectrometry. We then detailed the fragmentation pattern of dityrosine cross-linked Aß(1-16), using collision induced dissociation (CID), higher-energy collision induced dissociation (HCD), electron transfer dissociation (ETD), and electron capture dissociation (ECD). Application of these generic fragmentation rules of dityrosine cross-linked peptides allowed for the identification of dityrosine cross-links in peptides of Aß and α-synuclein generated in vitro by enzymatic peroxidation. We report, for the first time, the dityrosine cross-linked residues in human hemoglobin and α-synuclein under oxidative conditions. Together these tools open up the potential for automated analysis of this naturally occurring post-translation modification in neurodegenerative diseases as well as other pathological conditions.

Correlation of Phosphorus Cross-Linking to Hydration Rates in Sodium Starch Glycolate Tablet Disintegrants Using MRI.

Understanding the behavior of tablet disintegrants is valuable in the development of pharmaceutical solid dosage formulations. In this study, high-resolution magnetic resonance imaging has been used to understand the hydration behavior of a series of commercial sodium starch glycolate (SSG) samples, providing robust estimates of tablet disintegration rate that could be correlated with physicochemical properties of the SSGs, such as the extent of phosphorus (P) cross-linking as obtained from infra-red spectroscopy. Furthermore, elemental analysis together with powder X-ray diffraction has been used to quantify the presence of carboxymethyl groups and salt impurities, which also contribute to the disintegration behavior. The utility of Fast Low Angle SHot magnetic resonance imaging has been demonstrated as an approach to rapidly acquire approximations of the volume of a disintegrating tablet and, together with a robust voxel analysis routine, extract tablet disintegration rates. In this manner, a complete characterization of a series of SSG grades from different sources has been performed, showing the variability in their physicochemical properties and demonstrating a correlation between their disintegration rates and intrinsic characteristics. The insights obtained will be a valuable aid in the choice of disintegrant source as well as in managing SSG variability to ensure robustness of drug products containing SSG.

A biochemical study of the distribution of collagen and its crosslinks in knee ligaments and the patellar tendon.

PURPOSE: The purpose of this study was to investigate biochemical differences in collagen crosslinks from different locations within the ligaments and a tendon of the human knee. MATERIALS AND METHODS: The anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament (MCL), lateral collateral ligament (LCL), and patellar tendon (PT) were obtained from 24 cadavers (13 men and 11 women) whose average age at the time of death was 84.8 years. Ligaments and PT samples were obtained from the femoral and tibial insertions and the midsubstance. Hydroxyproline (Hyp) and collagen crosslinks, including pyridinoline (Pyr) and pentosidine (Pen), were compared among the different sites. RESULTS: The midsubstance Hyp concentration was greater than at the femoral and tibial insertions in the ACL (p = 0.00124 and 0.000255, respectively) and PCL (p = 0.00036 and 0.042, respectively). The Pyr:collagen ratio did not differ among sites in any of the ligaments or PT. The Pen:collagen ratio at the midsubstance was greater than at the femoral and tibial insertions in the ACL (p = 0.00022 and 0.00025, respectively) and LCL (p = 0.000081 and 0.000021, respectively) and was greater at the femoral insertion in the MCL (p = 0.00010). CONCLUSIONS: The mature collagen crosslink Pyr was not different in distribution in knee ligaments and the PT. Pen increased at the midsubstance ligaments and the PT. As increased Pen may represent ligament degeneration, this may indicate that degeneration may progress more rapidly at the midsubstance than at the insertion sites of a ligament.

A quantitative mass spectrometry-based approach for assessing the repair of 8-methoxypsoralen-induced DNA interstrand cross-links and monoadducts in mammalian cells.

Interstrand cross-links (ICLs) are highly toxic DNA lesions that block transcription and replication by preventing strand separation. ICL-inducing agents were among the earliest and are still the most widely used forms of chemotherapeutic drugs. Because of the repair of DNA ICLs, the therapeutic efficacy of the DNA cross-linking agents is often reduced by the development of chemoresistance in patients. Thus, it is very important to understand how various DNA ICLs are repaired. Such studies are currently hampered by the lack of an analytical method for monitoring directly the repair of DNA ICLs in cells. Here we report a high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method, together with the isotope dilution technique, for assessing the repair of 8-methoxypsoralen (8-MOP)-induced DNA ICLs, as well as monoadducts (MAs), in cultured mammalian cells. We found that, while there were substantial decreases in the levels of ICL and MAs in repair-competent cells 24 h after 8-MOP/UVA treatment, there was little repair of 8-MOP-ICLs and -MAs in xeroderma pigmentosum, complementation group A-deficient human skin fibroblasts and excision repair cross-complementing rodent repair deficiency, complementation group 1-deficient Chinese hamster ovary cells over a 24 h period. This result provided unequivocal evidence supporting the notion that the 8-MOP photoadducts are substrates for nucleotide excision repair in mammalian cells. This is one of the first few reports about the application of LC-MS/MS for assessing the repair of DNA ICLs. The analytical method developed here, when combined with genetic manipulation, will also facilitate the assessment of the roles of other DNA repair pathways in removing these DNA lesions, and the method can also be generally applicable for investigating the repair of other types of DNA ICLs in mammalian cells.

Tomato Allergy: Clinical Features and Usefulness of Current Routinely Available Diagnostic Methods

Introducción: El tomate contiene numerosos alérgenos cuya relevancia clínica, así como la utilidad de los métodos diagnósticos disponibles, están por definir. Objetivo: Evaluar la utilidad clínica de los métodos de diagnóstico en alergia a tomate. Métodos: Para ello se estudiaron 96 adultos con alergia alimentaria a vegetales y su reactividad frente a proteínas PR-10, profilina y LTP. La alergia a tomate fue definida por historia clínica y prueba cutánea (prick) positiva a tomate fresco. Se realizaron pruebas cutáneas con extracto comercial de tomate y determinación de IgE específica. Resultados: Un 36%, 8%, 28%, 18%, 8%, y 1% de los pacientes estaban sensibilizados a PR-10, profilina, ambas (PR-10 y profilina), LTP sola, LTP más PR-10 o profilina, y alérgenos genuinos del tomate respectivamente. 32/96 (33%) de los pacientes tenían alergia a tomate; alergia que se asocia a hipersensibilidad a profilinas (p< 0,001). La sensibilidad del prick fue buena en todos los subgrupos, pero la especificidad fue baja. La sensibilidad del ImmunoCAP fue aceptable en los reactores a profilina, pero muy pobre en reactores a PR-10. Los niveles de IgE no se asocian con la alergia al tomate en ningún subgrupo. De forma similar los niveles de IgE específica frente a abedul y melocotón no se asociaban con alergia a tomate en PR-10/profilina, o en reactores a LTP, respectivamente. Ambos test funcionaron bien en los alérgicos genuinos a tomate. Los niveles de IgE específica a tomate y abedul no estaban asociados en los pacientes monosensibles a PR-10, pero se correlacionaban en el grupo de profilinas (p < 0,005). Los niveles de IgE específica frente a tomate y abedul se correlacionaban en los pacientes alérgicos a LTP. Conclusiones: La alergia a tomate ocurre vía sensibilización a diferentes proteínas. El diagnóstico basado en componentes ayuda a definir los subgrupos clínicos con un riesgo diferente (AU)

Background: Tomato contains many allergens but their clinical relevance is poorly defined and the usefulness of available diagnostic methods is unknown. Objective: To assess the clinical usefulness of current diagnostic methods for tomato allergy. Methods: Ninety-six adults with plant food allergy were grouped based on their reactivity to PR-10, profilin, and lipid transfer protein (LTP). Tomato allergy was ascertained by history and a positive skin prick test (SPT) to fresh tomato. SPT with a commercial extract and immunoglobulin (Ig) E measurements were carried out. Results: In total, 36%, 8%, 28%, 18%, 8%, and 1% of patients were sensitized to PR-10, profilin, both PR-10 and profilin, LTP alone, LTP plus PR-10 or profilin, and genuine tomato allergens, respectively. Tomato allergy was detected in 32 (33%) of the 96 patients and was significantly associated with profilin hypersensitivity (P<.001). The sensitivity of SPT was good in all subgroups, but specificity was poor in many cases. ImmunoCAP sensitivity was acceptable in profilin reactors, but very poor in PR-10 reactors. IgE levels were not associated with tomato allergy in any of the subgroups. Similarly, birch and peach-specific IgE levels were not associated with tomato allergy in PR-10/profilin or in LTP reactors, respectively. Both SPT and ImmunoCAP worked well in the only patients with true tomato allergy. Birch- and tomato-specific IgE levels were not associated in patients monosensitized to PR-10, but they were correlated in profilin groups (P<.005). Peach- and tomato-specific IgE levels were correlated (P<.001) in LTP-allergic patients. Conclusions: Tomato allergy occurs via sensitization towards different proteins. Component-resolved diagnosis helps to define clinical subgroups with different risk levels (AU)

Nondestructive fluorescence-based quantification of threose-induced collagen cross-linking in bovine articular cartilage.

Extensive collagen cross-linking affects the mechanical competence of articular cartilage: it can make the cartilage stiffer and more brittle. The concentrations of the best known cross-links, pyridinoline and pentosidine, can be accurately determined by destructive high-performance liquid chromatography (HPLC). We explore a nondestructive evaluation of cross-linking by using the intrinsic fluorescence of the intact cartilage. Articular cartilage samples from bovine knee joints were incubated in threose solution for 40 and 100 h to increase the collagen cross-linking. Control samples without threose were also prepared. Excitation-emission matrices at wavelengths of 220 to 950 nm were acquired from the samples, and the pentosidine and pyridinoline cross-links and the collagen concentrations were determined using HPLC. After the threose treatment, pentosidine and lysyl pyridinole (LP) concentrations increased. The intrinsic fluorescence, excited below 350 nm, decreased and was related to pentosidine [r = -0.90, 240/325 nm (excitation/emission)] or LP (r = -0.85, 235/285 nm) concentrations. Due to overlapping, the changes in emission could not be linked specifically to the recorded cross-links. However, the fluorescence signal enabled a nondestructive optical estimate of changes in the pentosidine and LP cross-linking of intact articular cartilage.

Investigation of two-photon excited fluorescence increment via crosslinked bovine serum albumin.

The two-photon excited fluorescence (TPEF) increments of two dyes via bovine serum albumin (BSA) microstructures fabricated by the two-photon crosslinking technique were investigated. One is Rose Bengal (RB) with a high non-radiative decay rate, while the other is Eosin Y with a low non-radiative decay rate. Experimental results demonstrate that the quantum yield and lifetime of RB are both augmented via crosslinked BSA microstructures. Compared with theoretical analysis, this result indicates that the non-radiative decay rate of RB is decreased; hence, the quenched effect induced by BSA solution is suppressed. However, the fluorescence lifetime of Eosin Y is acutely abated despite the augmented quantum yield for the two-photon crosslinking processing from BSA solution. This result deduces that the radiative decay rate increased. Furthermore, the increased TPEF intensity and lifetime of RB correlated with the concentration of fabricated crosslinked BSA microstructures through pulse selection of the employed femtosecond laser is demonstrated and capable of developing a zone-plate-like BSA microstructure.