Chondrocyte death after drilling and articular screw insertion in a bovine model.

OBJECTIVE: Intra-articular screws are used for internal fixation of osteochondral fragments after fracture or osteochondritis dissecans. This causes cartilage injury potentially leading to chondrocyte death. We have visualised/quantified the hole and zone of cell death (ZCD) in cartilage after drilling/insertion of various articular screws. METHOD: Using an ex vivo bovine model with transmitted light and confocal laser scanning microscopy (CLSM), the holes and ZCD following drilling/insertion of articular screws (cortical screw, headless variable pitch metallic screw, headless variable pitch bioabsorbable screw) were evaluated. In situ chondrocyte death was determined by live/dead cell viability assay. An imaging/quantification protocol was developed to compare hole diameter and ZCD from drilling/insertion of screws into cartilage. The effect of saline irrigation during drilling on the ZCD was also quantified. RESULTS: Screw insertion created holes in cartilage that were significantly (P ≤ 0.001) less than the diameters of the equipment used. With a 1.5 mm drill, a ZCD of 580.2 ± 124 µm was produced which increased to 637.0 ± 44 µm following insertion of a 2 mm cortical screw although this was not significant (P > 0.05). The ZCD from insertion of the variable pitch headless screws (diam. 3.5 mm) was lower for the metallic compared to the bioabsorbable design (800.9 ± 159 vs 1,236.4 ± 212 µm, respectively; P < 0.01). The ZCD from drilling was reduced ∼50% (P < 0.001) by saline irrigation. CONCLUSIONS: Cartilage injury during intra-articular screw fixation caused a ZCD around the hole irrespective of screw design. Saline irrigation significantly reduced the ZCD from drilling into cartilage.

Rapid in situ chondrocyte death induced by Staphylococcus aureus toxins in a bovine cartilage explant model of septic arthritis.

OBJECTIVE: To assess in situ chondrocyte viability following exposure to a laboratory strain and clinical isolates of Staphylococcus aureus. METHODS: Bovine cartilage explants were cultured in the presence of S. aureus 8325-4 (laboratory strain), clinical S. aureus isolates or non-infected culture medium of pH values 7.4, 6.4 and 5.4. All clinical isolates were isolated from the joint aspirates of patients presenting with S. aureus-induced septic arthritis (SA). At designated time points, in situ chondrocyte viability was assessed within defined regions-of-interest in the axial and coronal plane following live- and dead-cell image acquisition using the fluorescent probes 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI), respectively, and confocal laser-scanning microscopy (CLSM). Cartilage water content, following S. aureus 8325-4 exposure, was obtained by measuring cartilage wet and dry weights. RESULTS: S. aureus 8325-4 and clinical S. aureus isolates rapidly reduced in situ chondrocyte viability (>45% chondrocyte death at 40 h). The increased acidity, observed during bacterial culture, had a minimal effect on chondrocyte viability. Chondrocyte death commenced within the superficial zone (SZ) and rapidly progressed to the deep zone (DZ). Simultaneous exposure of SZ and DZ chondrocytes to S. aureus 8325-4 toxins found SZ chondrocytes to be more susceptible to the toxins than DZ chondrocytes. Cartilage water content was not significantly altered compared to non-infected controls. CONCLUSIONS: Toxins released by S. aureus have a rapid and fatal action on in situ chondrocytes in this experimental model of SA. These data advocate the prompt and thorough removal of bacteria and their toxins during the treatment of SA.

Apollo lunar module engine exhaust products.

Organic combustion products generated by the lunar module descent engine, which burns a 1:1 mixture of unsymmetrical dimethylhydrazine fuel and nitrogen tetroxide oxidizer, have been analyzed. The major gaseous combustion products found were ammonia, water, carbon monoxide, nitrous oxide, oxygen, carbon dioxide, and nitric oxide. The minor products were acetylene, hydrogen cyanide, ethylene, formaldehyde, propadiene, ketene, cyanous acid, hydrazoic acid, various methylamines, acetaldehyde, methyl nitrite, formic acid, nitrous acid, butadiyne, nitrilohydrazines, nitromethane, and nitrosohydrazines with other oxidized derivatives of unsymmetrical dimethylhydrazine and hydrazine. The ion intensities of the various species in all mass spectra were estimated as the following concentrations: the gases (NH(3), H(2)O, CO, NO, O(2), CO(2), and NO(2)), 87.7 percent; compounds of C, H, and O, 6.0 percent; and compounds of C, H, and N (with traces of O), 5.8 percent.

Tranexamic acid toxicity in human periarticular tissues.

OBJECTIVES: Tranexamic acid (TXA) is an anti-fibrinolytic medication commonly used to reduce perioperative bleeding. Increasingly, topical administration as an intra-articular injection or perioperative wash is being administered during surgery. Adult soft tissues have a poor regenerative capacity and therefore damage to these tissues can be harmful to the patient. This study investigated the effects of TXA on human periarticular tissues and primary cell cultures using clinically relevant concentrations. METHODS: Tendon, synovium, and cartilage obtained from routine orthopaedic surgeries were used for ex vivo and in vitro studies using various concentrations of TXA. The in vitro effect of TXA on primary cultured tenocytes, fibroblast-like synoviocytes, and chondrocytes was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assays, fluorescent microscopy, and multi-protein apoptotic arrays for cell death. RESULTS: There was a significant (p < 0.01) increase in cell death within all tissue explants treated with 100 mg/ml TXA. MTT assays revealed a significant (p < 0.05) decrease in cell viability in all tissues following treatment with 50 mg/ml or 100 mg/ml of TXA within four hours. There was a significant (p < 0.05) increase in cell apoptosis after one hour of exposure to TXA (100 mg/ml) in all tissues. CONCLUSION: The current study demonstrates that TXA caused significant periarticular tissue toxicity ex vivo and in vitro at commonly used clinical concentrations.Cite this article: M. McLean, K. McCall, I. D. M. Smith, M. Blyth, S. M. Kitson, L. A. N. Crowe, W. J. Leach, B. P. Rooney, S. J. Spencer, M. Mullen, J. L. Campton, I. B. McInnes, M. Akbar, N. L. Millar. Tranexamic acid toxicity in human periarticular tissues. Bone Joint Res 2019;8:11-18. DOI: 10.1302/2046-3758.81.BJR-2018-0181.R1.

A potential key role for alpha-haemolysin of Staphylococcus aureus in mediating chondrocyte death in septic arthritis.

OBJECTIVES: Staphylococcus aureus (S. aureus) is the most commonly implicated organism in septic arthritis, a condition that may be highly destructive to articular cartilage. Previous studies investigating laboratory and clinical strains of S. aureus have demonstrated that potent toxins induced significant chondrocyte death, although the precise toxin or toxins that were involved was unknown. In this study, we used isogenic S. aureus mutants to assess the influence of alpha (Hla)-, beta (Hlb)-, and gamma (Hlg)-haemolysins, toxins considered important for the destruction of host tissue, on in situ bovine chondrocyte viability. METHODS: Bovine cartilage explants were cultured with isogenic S. aureus mutants and/or their culture supernatants. Chondrocyte viability was then assessed within defined regions of interest in the axial and coronal plane following live- and dead-cell imaging using the fluorescent probes 5-chloromethylfluorescein diacetate and propidium iodide, respectively, and confocal laser-scanning microscopy. RESULTS: Hla-producing mutants caused substantial chondrocyte death compared with the toxin-deficient control (Hla-Hlb-Hlg-), whilst mutants producing Hlb and Hlg in the absence of Hla induced minimal chondrocyte death. Coronal studies established that Hla-induced chondrocyte death started in the superficial zone of cartilage and spread to deeper layers, whereas Hlb and Hlg toxins were without significant effect. CONCLUSION: This study identified Hla as a highly potent S. aureus toxin that caused rapid chondrocyte death in bovine cartilage, with other toxins or metabolic products produced by the bacteria playing a minor role. The identification of Hla in mediating chondrocyte death may assist in the development of therapeutic strategies aimed at reducing the extent of cartilage damage during and after an episode of septic arthritis.Cite this article: I. D. M. Smith, K. M. Milto, C. J. Doherty, S. G. B. Amyes, A. H. R. W. Simpson, A. C. Hall. A potential key role for alpha-haemolysin of Staphylococcus aureus in mediating chondrocyte death in septic arthritis. Bone Joint Res 2018;7:457-467. DOI: 10.1302/2046-3758.77.BJR-2017-0165.R1.

Oestrone, oestradiol-17beta and oestriol levels in human foetal plasma during gestation and at term.

PIP: Marked rises in both unconjugated and sulphoconjugated estrone, estradiol-17-beta and estriol were observed in human fetal plasma between midgestation and term. Significant arterio-venous differences were found in the umbilical cord plasma. No consistent arterio-venous differences were found in the umbilical cord plasma. This indicates that all 3 estrogens are secreted from the placenta into the fetal circulation in the unconjugated form. Mean unconjugated estrogen (estrone + estradiol-17-beta + estriol) levels rose from 22.7 ng/ml at 17-20 weeks of gestation to 108.9 ng/ml at term in umbilical venous plasma and from 4.3 ng/ml to 23.3 ng/ml in umbilical arterial plasma. This represents a secretion rate of approximately 30 mg estrogen/day into the umbilical vein at term. Mean estrogen sulphate levels rose from 128 ng/ml to 313 ng/ml in the cord plasma during the same period. Of the 3 estrogens measured, estriol was quantitatively the major estrogen in fetal plasma. It consistently represented about 78% of the unconjugated fraction and 95% of the sulphate fraction at all stages of gestation. The method of delivery did not have a significant effect on the estrogen levels in uncomplicated pregnancies. Similar estrogen levels were found in fetal heart blood after either hysterotomy at spontaneous abortion at 16-20 weeks of gestation, and no significant differences were found for estrogen levels in cord plasma after elective Caesarean section at 38-39 weeks when compared with estrogen levels after normal delivery at term. A significant rise in fetal unconjugated estrogens at a time when fetal corticosteroids are increasing may be of physiological importance for fetal maturation in women.^ieng

Contralateral turning caused by metabotropic glutamate receptor stimulation in the dorsal striatum is reversed by MCPG, TTX, and cis-flupenthixol.

Recent evidence suggests an involvement of metabotropic glutamate receptors in the physiology of the striatum. In this study, rotation was recorded in an automated rotometer for 20 min following dorsal striatal injections (0.5 microliter) in cannulated rats. The metabotropic agonist 1-aminocyclopentane-trans-1, 3-dicarboxylic acid (1S,3R-ACPD) caused dose-dependent contralateral rotation. Turning caused by 500 microM 1S,3R-ACPD was reversed by coinjections of the metabotropic antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG, 1 mM) and by tetrodotoxin (100 microM). Injections of MCPG alone (10 microM, 100 microM, 1 mM) failed to elicit turning. Increasing doses of the dopamine antagonist cis-flupenthixol also reversed 1S,3R-ACPD-induced rotation. Thus unilateral striatal metabotropic glutamate receptor stimulation can cause receptor-specific rotation that may result from an increase in neural activity, and is dependent on intact dopamine neurotransmission.

Glutamate receptor agonist injections into the dorsal striatum cause contralateral turning in the rat: involvement of kainate and AMPA receptors.

Unilateral stimulation of glutamate receptors in the dorsal striatum of intact rats resulted in contralateral turning. Turning behavior was recorded for 20 min following unilateral intrastriatal injections (0.5 microliter) in chronically cannulated rats. Kainate injections caused a dose-dependent increase in contralateral rotation that was blocked by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the action potential blocker tetrodotoxin, and by increasing doses of the dopamine receptor antagonist cis-flupenthixol. Injections of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) also caused rotation that was blocked with co-injections of CNQX, tetrodotoxin or cis-flupenthixol. Neither CNQX nor tetrodotoxin injected alone caused turning. This effect is dopamine-dependent, and may result from a kainate or AMPA-induced increase in dopamine release. Glutamate receptor agonist injections into the striatum may cause contralateral turning by degrading information in ascending cortical projections and may further influence locomotion via basal ganglia output nuclei projections to the brainstem.

Rotational bias in intact rats following intrastriatal injections of dopaminergic drugs.

Following unilateral 6-OHDA lesions of the striatum, systemic dopamine agonists produce rotation due to receptor supersensitivity. Rotation following intrastriatal dopamine agonists in intact rats also has been reported, although these studies are few and contradictory. Dorsal striatal injection (0.5 microl) of the direct dopamine agonist apomorphine failed to caused rotation. In addition, neither the D1 agonist SKF 81297, the cAMP analogue Sp-cAMPS, nor the D2 agonist quinpirole affected rotation. In contrast, the dopamine releaser amphetamine (1.1, 10.9, 108.7 mM) caused significant contralateral rotation. This effect was reversed by coinjection of the D1 antagonist SCH 23390 (3.1 mM) but not by the D2 antagonist eticlopride. Rotation was also reversed by TTX coinjections (100 microM) but not by the NMDA antagonist AP7 or the kainate/AMPA antagonist CNQX. Thus, direct dopamine agonists in the striatum failed to cause behavioral asymmetry, whereas amphetamine induced contralateral rotation. This effect is mediated primarily by D1 receptors and requires concurrent neuronal activation that appears to be independent of glutamate receptor stimulation. These results are consistent with studies of Fos induction in normosensitive animals following dopamine agonists and are discussed in terms of changes in basal ganglia output pathway activity.

Hair testing for "ecstasy" (MDMA) in volunteer Scottish drug users.

The aim of the study was to compare self reported "ecstasy" use with the results of the analysis of hair harvested from the same users. Subjects were recruited by multisite chain-referral sampling within the 1994-95 "dance scene" in Glasgow. One hundred subjects donated hair after completing a lengthy interviewer-administered questionnaire. Overall gross concordance between self reported "ecstasy" use and discovery of MDMA (or related compounds) in analysed hair did not surpass 59%, and no relationship had a Cohen's kappa of more than 0.08. Within the positive concordant dataset (n = 52), scatter was considerable, with no correlation being significant, and none more strongly positive than -0.0518. The results presented here indicate that, as far as MDMA is concerned, if judged by self-report, hair does not reach a level of apparent accuracy that would permit its use as a general population estimator. However, hair testing is probably more reliable than self-report, and its accuracy could be verified independently if large-scale inter- and intra-laboratory comparative research is conducted.

Hair analysis: self-reported use of "speed" and "ecstasy" compared with laboratory findings.

Drug use histories were collected from 100 subjects recruited from the "dance scene" in and around Glasgow, Scotland. In addition, each subject donated a hair sample which was analyzed by gas chromatography/mass spectrometry (GC/MS) for amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MD MA) and 3,4-methylenedioxyethylamphetamine (MDEA). The hair samples were analyzed in two 6 cm segments or in full, ranging from 1.5 to 12 cm depending on the length of the hair. Approximately 10 mg of hair was ground to a fine powder before treatment with beta-glucuronidase/aryl sulfatase. A solid-phase extraction procedure was carried out followed by derivatization with pentafluoropropionic anhydride (PFPA). All extracts were analyzed by gas chromatography/mass spectrometry (GC/MS). Of the 139 segments analyzed, 77 (52.5%) were positive for at least one of the five amphetamines. The drug concentrations found in the hair were compared with the self-reported drug histories. A concordance of greater than 50% was found between the self-report data and levels detected in hair. However, no correlation was found between the reported number of "ecstasy" tablets consumed and the drug levels detected in hair. An increase in the average drug levels measured was observed from low to high use (number of "ecstasy" tablets/month). A large number of false negatives and a low number of false positives were observed.