Comparison of mechanical properties of various suture repair techniques in a partially lacerated tendon

The technique of surgical repair for zone two flexor tendon injuries has been debated extensively throughout the years, yet adhesion formation, suture rupture, and suture locking on the pulley edge remain possible consequences of a poor repair. The partially lacerated tendon is especially challenging to treat since there can be justification for not intervening surgically. In a partial laceration canine model we measured failure load and suture gap formation for tendons repaired with the Lee, modified four-strand Savage, Kessler, modified Kessler, and Augmented Becker core suture techniques and with a simple running peripheral suture. The modified Kessler (106.3 N, SD 18.8 N) and modified Savage (108.2 N, SD 19.9 N) repair techniques had a significantly higher failure load than the Lee (85.0 N, SD 20.6 N) suture method (P < 0.05), while there were no differences among the other techniques. There were no significant differences in resistance to gap formation among the repair techniques, with the mean values ranging from 38.9 N/mm (SD 15.7 N/mm) using the simple running suture to 53.2 N/mm (SD 25.8 N/mm) with the Kessler repair. The mean load to produce a 1.5 mm repair site gap ranged from 71.1 N (SD 21.5 N) in the Lee repair to 91.3 N (SD 22.2 N) in the Augmented Becker repair although there were no significant differences among repair methods. All repair methods were much weaker than tendons left unrepaired (184.7 N, SD 41.3 N).     

Biotransformation of fluorobiphenyl by Cunninghamella elegans

The fungus Cunninghamella elegans is a useful model of human catabolism of xenobiotics. In this paper, the biotransformation of fluorinated biphenyls by C. elegans was investigated by analysis of the culture supernatants with a variety of analytical techniques. 4-Fluorobiphenyl was principally transformed to 4-fluoro-4′-hydroxybiphenyl, but other mono- and dihydroxylated compounds were detected in organic extracts by gas chromatography–mass spectrometry. Additionally, fluorinated water-soluble products were detected by ¹⁹F NMR and were identified as sulphate and β-glucuronide conjugates. Other fluorobiphenyls (2-fluoro-, 4,4′-difluoro- and 2,3,4,5,6-pentafluoro-biphenyl) were catabolised by C. elegans, yielding mono- and dihydroxylated products, but phase II metabolites were detected from 4,4′-difluorobiphenyl only.     

Filamentous fungal biofilm for production of human drug metabolites

In drug development, access to drug metabolites is essential for assessment of toxicity and pharmacokinetic studies. Metabolites are usually acquired via chemical synthesis, although biological production is potentially more efficient with fewer waste management issues. A significant problem with the biological approach is the effective half-life of the biocatalyst, which can be resolved by immobilisation. The fungus Cunninghamella elegans is well established as a model of mammalian metabolism, although it has not yet been used to produce metabolites on a large scale. Here, we describe immobilisation of C. elegans as a biofilm, which can transform drugs to important human metabolites. The biofilm was cultivated on hydrophilic microtiter plates and in shake flasks containing a steel spring in contact with the glass. Fluorescence and confocal scanning laser microscopy revealed that the biofilm was composed of a dense network of hyphae, and biochemical analysis demonstrated that the matrix was predominantly polysaccharide. The medium composition was crucial for both biofilm formation and biotransformation of flurbiprofen. In shake flasks, the biofilm transformed 86 % of the flurbiprofen added to hydroxylated metabolites within 24 h, which was slightly more than planktonic cultures (76 %). The biofilm had a longer effective lifetime than the planktonic cells, which underwent lysis after 2?×?72 h cycles, and diluting the Sabouraud dextrose broth enabled the thickness of the biofilm to be controlled while retaining transformation efficiency. Thus, C. elegans biofilm has the potential to be applied as a robust biocatalyst for the production of human drug metabolites required for drug development.     

Biotransformation of Flurbiprofen by Cunninghamella Species

The biotransformation of the fluorinated anti-inflammatory drug flurbiprofen was investigated in Cunninghamella spp. Mono- and dihydroxylated metabolites were detected using gas chromatography-mass spectrometry and fluorine-19 nuclear magnetic resonance spectroscopy, and the major metabolite 4′-hydroxyflurbiprofen was isolated by preparative high-pressure liquid chromatography (HPLC). Cunninghamella elegans DSM 1908 and C. blakesleeana DSM 1906 also produced a phase II (conjugated) metabolite, which was identified as the sulfated drug via deconjugation experiments.One of the objectives of the recent European Union legislation governing the testing and evaluation of chemicals, REACH (Regulation, Evaluation, Authorisation and Restriction of Chemicals), is to further reduce the need for animals in the testing process. Some microorganisms, such as the zygomycete fungus Cunninghamella and actinomycetes bacteria, have been shown to metabolize xenobiotic compounds in a fashion analogous to that of mammals (3, 5, 11, 17). It was suggested over 3 decades ago that microorganisms had potential as models of mammalian metabolism (16), although there are concerns about their predictive value (8). Nevertheless, certain microorganisms can be applied to the generation of useful quantities of drug metabolic intermediates (13), which is more desirable than isolation of these compounds from dosed animals, and avoids the concerns often associated with chemical synthesis, such as the use of toxic reagents and harsh reaction conditions.Owing to the desirable physicochemical properties of the fluorine atom (small Van der Waals radius, electronegativity, and strength of the carbon-fluorine bond), approximately 25% of drugs either currently on the market or in the pipeline are fluorinated (12). One such example is flurbiprofen [(RS)-2-(2-fluoro-4-biphenyl)propionic acid], which is a nonsteroidal anti-inflammatory drug (NSAID) used in the treatment of inflammation caused by arthritis. In humans it is transformed to the phase I (oxidative) metabolites 4′-hydroxyflurbiprofen, 3′,4′-dihydroxyflurbiprofen, and 3′-hydroxy,4′-methoxyflurbiprofen; glucuronide and sulfate conjugates (phase II metabolites) have also been detected (9, 15). In equine urine additional hydroxylated and methoxylated metabolites were detected (20). Tracy et al. (18) demonstrated that only one cytochrome P450 isoform (2C9) is involved in the oxidation of flurbiprofen, which makes the drug a potentially useful in vivo probe for this particular isoform. Despite the prevalence of fluorinated drugs, only a few investigations have been undertaken to determine the microbial biotransformation of these compounds (7, 21). Here we describe the biotransformation of flurbiprofen by Cunninghamella species and the determination of the metabolites by nuclear magnetic resonance (NMR) spectroscopy (¹H and ¹⁹F), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC).Three species of Cunninghamella were selected for the biotransformation experiments: C. elegans (strains DSM 1908, DSM 8217, and DSM 63299), C. echinulata DSM 1905, and C. blakesleeana DSM 1906. The fungi were grown on Sabouraud dextrose agar plates (Sigma) for 5 days at 26°C before being homogenized in 100 ml of sterile saline solution. The homogenate (10%, vol/vol) was used to inoculate 50 ml of fresh Sabouraud dextrose broth in 250-ml Erlenmeyer flasks, which were incubated at 28°C with shaking at 150 rpm. Following previously established procedures (2), 5 mg of flurbiprofen (Sigma) dissolved in dimethyl formamide (20 μl) was added to the cultures after 72 h, and the incubation was continued up to a further 120 h. Control experiments were conducted in the absence of either flurbiprofen or fungus. The cultures (supernatant and cells) were sonicated on ice (Sonicator U200S control; IKA Labortechnik) for 5 min at 50% amplitude, with intervals of 30 s after each minute to prevent overheating. The sonicates were centrifuged, the supernatant was extracted with 50 ml of ethyl acetate, and the extracts were evaporated to dryness.     

Temporal response of canine flexor tendon to limb suspension

Tendon disuse, or stress deprivation, frequently accompanies clinical disorders and treatments, yet the metabolism of tendons subject to stress deprivation has rarely been investigated systematically. The effects of stress deprivation on canine flexor tendon were investigated in this study. One adult canine forepaw was suspended for 21 or 42 days. Control forepaws were collected from dogs that had no intervention on their limbs and paws. The expression of collagen I and III was not significantly altered in the tendons disused for 21 days but was significantly decreased at 42 days (P < 0.03). The expression of collagen II, aggrecan, decorin, and fibronectin was significantly decreased in the tendons in the suspended limbs at 21 days (P < 0.002) and further reduced at 42 days. With stress deprivation, the expression of matrix metalloproteinase 2 (MMP2) was significantly increased (P < 0.004) at 21 and 42 days. The expression of MMP3 was significantly decreased at 21 and 42 days (P < 0.03). The expression of MMP13 was not altered with stress deprivation at 21 and 42 days. The expression of MMP14 was significantly increased at 21 days (P = 0.0015) and returned to the control level at 42 days. Tissue inhibitor of metalloproteinase 1 (TIMP1) expression was decreased after the limbs were suspended for 42 days (P = 0.0043), but not 21 days. However, TIMP2 expression was not significantly different from control at 21 or 42 days. Furthermore, the cross-sectional area of the stress-deprived tendons at 42 days was decreased compared with the control group (P < 0.01). The intervention method in this study did not result in any alteration of stiffness of the tendon. Our study demonstrated that stress deprivation decreases the anabolic process and increases the catabolic process of extracellular matrix in flexor tendon.     

The P2Y4 receptor forms homo-oligomeric complexes in several CNS and PNS neuronal cells

It is well established that several cell surface receptors interact with each other to form dimers and oligomers, which are essential for their activation. Since little is known about the quaternary structure of P2Y receptors, in the present work, we investigated the expression of the G-protein-coupled P2Y₄ subunit as monomeric or higher-order complex protein. We examined both endogenously expressed P2Y₄ subtype with the aid of specific anti-P2Y₄ antiserum, and heterologously transfected P2Y₄-tagged receptors with the use of antitag antibodies. In both cases, we found the P2Y₄ receptor displaying molecular masses corresponding to monomeric, dimeric and oligomeric structures. Experiments performed in the absence of reducing agents demonstrated that there is a strict correlation among the multiple protein bands and that the multimeric forms are at least partially assembled by disulphide bonds. The direct demonstration of P2Y₄ homodimerisation comes instead from co–transfection and differential co–immunoprecipitation experiments, with the use of differently tagged P2Y₄ receptors and antitag antibodies. The structural propensity of the P2Y₄ protein to form homo-oligomers may open the possibility of a novel regulatory mechanism of physiopathological functions for this and additional P2Y receptors.     

Analysis of the gliding pattern of the canine flexor digitorum profundus tendon through the A2 pulley

Friction between a tendon and its pulley was first quantified using the concept of the arc of contact. Studies of human tendons conformed closely to a theoretical nylon cable/nylon rod model. However, we observed differences in measured friction that depended on the direction of motion in the canine model. We hypothesized that fibrocartilaginous nodules in the tendon affected the measurements and attempted to develop a theoretical model to explain the observations we made. Two force transducers were connected to each end of the canine flexor digitorum profundus tendon and the forces were recorded when it was moved through the A2 pulley toward a direction of flexion by an actuator and then reversed a direction toward extension. The changes of a force as a function of tendon excursion were evaluated in 20 canine paws. A bead cable/rod model was developed to simulate the canine tendon-pulley complex. To interpret the results, a free-body diagram was developed. The two prominent fibrocartilaginous nodules in the tendon were found to be responsible for deviation from a theoretical nylon cable gliding around the rod model, in a fashion analogous to the effect of the patella on the quadriceps mechanism. A bead cable/rod model qualitatively reproduced the findings observed in the canine tendon-pulley complex. Frictional coefficient of the canine flexor tendon-pulley was 0.016+/-0.005. After accounting for the effect created by the geometry of two fibrocartilaginous nodules within the tendon, calculation of frictional force in the canine tendon was possible.