See-saw rocking: an in vitro model for mechanotransduction research.

In vitro mechanotransduction studies, uncovering the basic science of the response of cells to mechanical forces, are essential for progress in tissue engineering and its clinical application. Many varying investigations have described a multitude of cell responses; however, as the precise nature and magnitude of the stresses applied are infrequently reported and rarely validated, the experiments are often not comparable, limiting research progress. This paper provides physical and biological validation of a widely available fluid stimulation device, a see-saw rocker, as an in vitro model for cyclic fluid shear stress mechanotransduction. This allows linkage between precisely characterized stimuli and cell monolayer response in a convenient six-well plate format. Models of one well were discretized and analysed extensively using computational fluid dynamics to generate convergent, stable and consistent predictions of the cyclic fluid velocity vectors at a rocking frequency of 0.5 Hz, accounting for the free surface. Validation was provided by comparison with flow velocities measured experimentally using particle image velocimetry. Qualitative flow behaviour was matched and quantitative analysis showed agreement at representative locations and time points. Maximum shear stress of 0.22 Pa was estimated near the well edge, and time-average shear stress ranged between 0.029 and 0.068 Pa. Human tenocytes stimulated using the system showed significant increases in collagen and GAG secretion at 2 and 7 day time points. This in vitro model for mechanotransduction provides a versatile, flexible and inexpensive method for the fluid shear stress impact on biological cells to be studied.

An ink surgical marker pen is damaging to tendon cells.

OBJECTIVES: Surgical marking during tendon surgery is often used for technical and teaching purposes. This study investigates the effect of a gentian violet ink marker pen, a common surgical marker, on the viability of the tissue and cells of tendon. METHODS: In vitro cell and tissue methods were used to test the viability of human hamstring explants and the migrating tenocytes in the presence of the gentian violet ink. RESULTS: The outcome of this study was that a constituent of the surgical marker pen causes cell and tissue death in culture, implying the same would occur in vivo. CONCLUSIONS: This is a cause for concern when marking tendon during surgical procedures, as it may compromise healing and repair and potentially contribute to a poor outcome. The authors suggest that an alternative surgical marking procedure should be found, or that all marker pens should undergo testing on human tendon tissue in vitro prior to use.

A systematic review of the histological and molecular changes in rotator cuff disease.

INTRODUCTION: The pathogenesis of rotator cuff disease (RCD) is complex and not fully understood. This systematic review set out to summarise the histological and molecular changes that occur throughout the spectrum of RCD. METHODS: We conducted a systematic review of the scientific literature with specific inclusion and exclusion criteria. RESULTS: A total of 101 studies met the inclusion criteria: 92 studies used human subjects exclusively, seven used animal overuse models, and the remaining two studies involved both humans and an animal overuse model. A total of 58 studies analysed supraspinatus tendon exclusively, 16 analysed subacromial bursal tissue exclusively, while the other studies analysed other tissue or varying combinations of tissue types including joint fluid and muscle. The molecular biomarkers that were altered in RCD included matrix substances, growth factors, enzymes and other proteins including certain neuropeptides. CONCLUSIONS: The pathogenesis of RCD is being slowly unravelled as a result of the significant recent advances in molecular medicine. Future research aimed at further unlocking these key molecular processes will be pivotal in developing new surgical interventions both in terms of the diagnosis and treatment of RCD.

Characterization of the metal-binding sites of the beta-lactamase from Bacteroides fragilis.

In an effort to better understand the structure and function of the metallo-beta-lactamase from Bacteroides fragilis, spectroscopic and metal-binding studies were performed on the native, metal-substituted, and mutant forms of the enzyme. Atomic absorption studies demonstrate that the native B. fragilis enzyme tightly binds 2 mol of Zn(II) and, along with mutagenesis studies, that the presence of both metal ions is required for full catalytic activity. EPR spectroscopy was used to confirm that the Co(II)-substituted beta-lactamase binds 2 mol of Co(II) per mole of enzyme, that the two Co(II)'s are highspin and probably uncoupled, with apparent g values of 6.5, 4.2, and 2.0, and that the coordination number of the Co(II) is 5 or 6. This number of ligands for the Co(II)-substituted enzyme is confirmed by UV-Vis spectra, which demonstrate the presence of very weak d-d transitions between 550 and 650 nm (epsilon approximately 30 M-1.cm-1) and an intense feature at 320 nm (epsilon approximately 1570 M-1.cm-1). The latter is assigned to a cysteine sulfur to Co(II) ligand-to-metal charge transfer band, and this assignment is confirmed by the disappearance of this band in the UV-Vis spectrum of a Co(II)-substituted C168S mutant. H NMR studies on the Co(II)-substituted enzyme suggest the presence of three histidine ligands bound to Co(II). Taken together, these studies support the sequence comparison study of Rasmussen et al., in which there is a catalytic metal-binding site with three histidines and one cysteine (C168). The remaining ligands are postulated to be water molecules involved in catalysis. Mutagenesis studies, in combination with activity assays and metal-binding studies, have been used to identify Asp61, Asp90, Asp152, and Asp183 as possible ligands to the second metal-binding site, with Asp90 and Asp152 having a pronounced effect on kcat. These results are discussed in light of the recent crystal structure of the metallo-beta-lactamase from B. cereus.

The acuityscope: a resolution test target projection ophthalmoscope.

The Acuityscope projects an image of a three bar resolution test target onto the patient's retina, where it is viewed and read by an observer. The test target consists of a series of three bar targets varying in size on a logarithmic scale. Individual targets are identified in the projected image by Bailey-Lovie log visual acuity equivalents. The projected image includes only those target sizes which are useful for estimating visual acuity. The Acuityscope is used for assessing the quality of retinal image formation, and is thus able to differentiate between poor visual acuity due to image degradation, (e.g. cataract or corneal disease) and poor visual acuity due to image handling defects (e.g. retinal or optic nerve disease). The use of the instrument is relevant to both clinical situations (e.g. pre-operative assessment of opacification of optical media) and experimental situations (e.g. clinical trials).

The transparency of the lens: a comparison between diabetic and non-diabetic patients.

The transparency of the lens of 485 eyes of 266 healthy volunteers were compared with 143 eyes of 83 diabetic patients. All the patients had well controlled diabetes. The transparency of the lens was measured with the opacity lensmeter 701. The concept of the instrument is based on the measurement of scattered light. The result revealed a similar transparency of the lenses of diabetic patients in relation to the normal volunteers. There was only a slight, and statistically not significant trend in the older age group versus a greater opacity for diabetic patients.

Treatment of cataract in diabetics with and without retinopathy.

The results of cataract surgery in diabetic patients was reviewed. The visual outcome was good in the absence of retinopathy and was not significantly different from that of non-diabetic patients. Eyes with retinopathy achieved significantly worse visual results and the prognosis was related to the severity of retinopathy. Clinical cystoid macular oedema occurred significantly more frequently in eyes with retinopathy than without and there were significantly more eyes with retinopathy which became blind or partially sighted. The use of a Projectoscope for assessing the amount of degradation of an image was tested. A good correlation was found between the amount of blurring of the test target and the drop in visual acuity. The optimal type of cataract surgery is discussed and lines of management outlined.

ECHO-11 as a respiratory virus: quantitation of infection in man.

Through a series of controlled experiments in volunteers, quantitative aspects of infection, illness, and immunity to ECHO-11 virus were studied. ECHO-11 is a transmissable viral infection in man and equally infectious to the upper respiratory and the intestinal tracts. The rate of infection was directly related to the dose of virus exposure, but any infectious dose of virus produced illness in only about one-third of the infected subjects. The infectious dose for man varied over a billionfold range. Larger challenge doses caused no difference in the local symptoms at the portal of entry or in the peak severity of illness, but symptoms were more diverse and prolonged after a higher dose. Persons with asymptomatic infections became just as heavily infected as ill persons. In respiratory secretions from natural infection, the titer of infectious virus was found to be about 10(2) median infective doses in tissue culture (TCID(50))/ml. At this level, up to 40% of exposed contacts could be infected per milliliter of secretion. The observed rate of spread was 24%. This low-dose inoculum caused illness in 12% of volunteers but failed to elicit a significant antibody response in 93% or immunity to reinfection and another illness upon rechallenge. Larger doses of virus produced a longer excretion of virus and a significant increase in serum-neutralizing antibody. Nasal antibody was infrequently found. The principal effect of antibody was to decrease virus excretion and to shorten illness; it reduced the rate but did not prevent infection. Attempts to produce an asymptomatic enteric infection which would induce immunity failed.The characteristics of respiratory transmission with mild disease, recurrent infection, and illness without a detectable antibody response or solid immunity to reinfection, satisfy the epidemiologic conditions to establish ECHO-11 virus as one of the causes of the common cold.