Dependence of crack shape in loaded articular cartilage on the collagenous structure.

Cartilage cracks disrupt tissue mechanics, alter cell mechanobiology, and often trigger tissue degeneration. Yet, some tissue cracks heal spontaneously. A primary factor determining the fate of tissue cracks is the compression-induced mechanics, specifically whether a crack opens or closes when loaded. Crack deformation is thought to be affected by tissue structure, which can be probed by quantitative polarized light microscopy (PLM). It is unclear how the PLM measures are related to deformed crack morphology. Here, we investigated the relationship between PLM-derived cartilage structure and mechanical behavior of tissue cracks by testing if PLM-derived structural measures correlated with crack morphology in mechanically indented cartilages. METHODS: Knee joint cartilages harvested from mature and immature animals were used for their distinct collagenous fibrous structure and composition. The cartilages were cut through thickness, indented over the cracked region, and processed histologically. Sample-specific birefringence was quantified as two-dimensional (2D) maps of azimuth and retardance, two measures related to local orientation and degree of alignment of the collagen fibers, respectively. The shape of mechanically indented tissue cracks, measured as depth-dependent crack opening, were compared with azimuth, retardance, or "PLM index," a new parameter derived by combining azimuth and retardance. RESULTS: Of the three parameters, only the PLM index consistently correlated with the crack shape in immature and mature tissues. CONCLUSION: In conclusion, we identified the relative roles of azimuth and retardance on the deformation of tissue cracks, with azimuth playing the dominant role. The applicability of the PLM index should be tested in future studies using naturally-occurring tissue cracks.

Deformation behaviors and mechanical impairments of tissue cracks in immature and mature cartilages.

Degeneration of articular cartilage is often triggered by a small tissue crack. As cartilage structure and composition change with age, the mechanics of cracked cartilage may depend on the tissue age, but this relationship is poorly understood. Here, we investigated cartilage mechanics and crack deformation in immature and mature cartilage exposed to a full-thickness tissue crack using indentation testing and histology, respectively. When a cut was introduced, tissue cracks opened wider in the mature cartilage compared to the immature cartilage. However, the opposite occurred upon mechanical indentation over the cracked region. Functionally, the immature-cracked cartilages stress-relaxed faster, experienced increased tissue strain, and had reduced instantaneous stiffness, compared to the mature-cracked cartilages. Taken together, mature cartilage appears to withstand surface cracks and maintains its mechanical properties better than immature cartilage and these superior properties can be explained by the structure of their collagen fibrous network.

Collagen fibres determine the crack morphology in articular cartilage.

Cracks in articular cartilage compromise tissue integrity and mechanical properties and lead to chondral lesions if untreated. An understanding of the mechanics of cracked cartilage may help in the prevention of cartilage deterioration and the development of tissue-engineered substitutes. The degeneration of cartilage in the presence of cracks may depend on the ultrastructure and composition of the tissue, which changes with aging, disease and habitual loading. It is unknown if the structural and compositional differences between immature and mature cartilage affect the mechanics of cartilage cracks, possibly predisposing one to a greater risk of degeneration than the other. We used a fibre-reinforced poro-viscoelastic swelling material model that accounts for large deformations and tension-compression non-linearity, and the finite element method to investigate the role of cartilage structure and composition on crack morphology and tissue mechanics. We demonstrate that the crack morphology predicted by our theoretical model agrees well with the histo-morphometric images of young and mature cracked cartilages under indentation loading. We also determined that the crack morphology was primarily dependent on collagen fibre orientation which differs as a function of cartilage depth and tissue maturity. The arcade-like collagen fibre orientation, first discussed by Benninghoff in his classical 1925 paper, appears to be beneficial for slowing the progression of tissue cracks by 'sealing' the crack and partially preserving fluid pressure during loading. Preservation of the natural load distribution between solid and fluid constituents of cartilage may be a key factor in slowing or preventing the propagation of tissue cracks and associated tissue matrix damage. STATEMENT OF SIGNIFICANCE: Cracks in articular cartilage can be detrimental to joint health if not treated, but it is not clear how they propagate and lead to tissue degradation. We used an advanced numerical model to determine the role of cartilage structure and composition on crack morphology under loading. Based on the structure and composition found in immature and mature cartilages, our model successfully predicts the crack morphology in these cartilages and determines that collagen fibre as the major determinant of crack morphology. The arcade-like Benninghoff collagen fibre orientation appears to be crucial in 'sealing' the tissue crack and preserves normal fluid-solid load distribution in cartilage. Inclusion of the arcade-like fibre orientation in tissue-engineered construct may help improve its integration within the host tissue.

Detection of new psychoactive substance use among emergency room patients: results from the Swedish STRIDA project.

The "STRIDA" project monitors the occurrence and trends of new psychoactive substances (NPS; "Internet drugs/designer drugs/legal highs") in Sweden, and collects information about their clinical symptoms, toxicity and associated health hazards. The initial results of the project documented a widespread use of many different NPS by mainly adolescents and young (age range 13-63 years, median 20), male (79%) adults, among cases of drug intoxications presenting at emergency departments and intensive care units across the country. The new substances were identified in samples of urine and blood by a multi-component LC-MS/MS method, and the severity of clinical symptoms were graded by the Poisoning Severity Score (PSS). Of the initial 189 samples submitted for laboratory investigation, 156 (83%) tested positive for at least one drug. Besides classical substances such as ethanol, cannabis and amphetamines, many NPS were detected comprising synthetic cannabinoid receptor agonists ("Spice"), piperazines, substituted phenethylamines, synthetic cathinones, hallucinogenic tryptamines, piperidines, opioid related substances, ketamine and related substances, and GABA analogues (in total more than 50 substances). About half of the cases were demonstrated to be multiple drug intoxications, sometimes making it hard to associate the clinical presentations with one specific substance. In conclusion, the STRIDA project has documented use of a broad variety of NPS among mainly young people all over Sweden.

Detectability of new psychoactive substances, 'legal highs', in CEDIA, EMIT, and KIMS immunochemical screening assays for drugs of abuse.

The increasing number of new psychoactive substances made available for recreational drug use has created a challenge for clinical toxicology and drug testing laboratories. As a consequence, the routine immunoassay drug testing may become less effective due to an increased occurrence of false negative and false positive screening results. This work aimed to extend the knowledge about analytical cross-reactivity of new substances in selected CEDIA, EMIT, and KIMS immunoassays for drugs-of-abuse screening. Urine standards were prepared by spiking blank urine with 45 new substances. Authentic urine samples from intoxication cases identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were also studied. Several new psychoactive substances were demonstrated to display cross-reactivity in the immunoassays. CEDIA Amphetamine/Ecstasy and EMIT d.a.u. Amphetamine Class tests showed the highest reactivity towards the new drugs, which was expected since many have amphetamine-like structure and activity. In the samples from authentic cases, five new substances displayed 100% detection rate in the CEDIA Amphetamine/Ecstasy test. In conclusion, cross-reactivity data in routine urine drug screening immunoassays for a number of new psychoactive substances not studied before were reported. In both spiked and authentic urine samples, some new substances showed significant cross-reactivity and are thus detectable in the routine screening methods.

Multicomponent LC-MS/MS screening method for detection of new psychoactive drugs, legal highs, in urine-experience from the Swedish population.

The advent of new not yet legally regulated psychoactive substances sold over the Internet has created a challenge for clinical toxicology and drug testing laboratories. The routine use of immunoassay screening may no longer be the optimal solution in many instances since the number of analytes covered is becoming insufficient. The aim of this work was to design, validate and apply a multi-component LC-MS/MS method suitable for screening of a large number of target analytes belonging to the class of new psychoactive substances - legal highs. The analytical method was using a five-fold dilution of urine with internal standard (pethidine-d5) and injection of 2µL. The chromatographic system was using a 1.7-µm 100mm×2.1mm Ethylene Bridged Hybrid (BEH) C18 column and gradient elution with a flow rate of 600µL/min. Solvent A consisted of 0.1% formic acid and Solvent B was 100% acetonitrile. The gradient elution application was designed to have a wide polarity coverage with total run time of 4.0min. The tandem mass spectrometer was using an electrospray interface and operated in positive mode. Selected reaction monitoring of two ion transitions was used for each of 26 analytes. Method validation demonstrated limited influence from urine matrix, linear response within the measuring range (0.1-10µg/mL), acceptable imprecision in quantification (CV<15%). Some analytes were found not to be stable in urine upon storage. The method was successfully applied in routine drug testing. A total of 87 positive samples with 100 analytical findings were found to contain O-desmethyl-cis-tramadol (mostly without mitragynine), methylenedioxypyrovalerone, 4-fluoroamphetamine, methoxetamine, desoxypipradol, 4-fluoromethcathinone, 5,6-methylenedioxy-2-aminoindane, 4-methylmethcathinone, 3-fluoromethcathinone, 4-hydroxy-N-methyl-N-ethyltryptamine, α-methylamino-butyrophenone and 4-methoxymethcathinone.