Treatment Depth Effects of Combined Magnetic Field Technology using a Commercial Bone Growth Stimulator

Lumbar spinal fusion is one of the more common spinal surgeries, and its use is on the rise. If the bone fails to fuse properly, then a pseudarthrosis or “false joint” develops and results in pain, instability, and disability. Since1974, three types of electrical stimulation technologies have been approved for clinical use to enhance spinal fusions. One such technology is inductive coupling, which includes combined magnetic fields (CMFs). The purpose of this study was to evaluate the effects of a CMF device known as the Donjoy (SpinaLogic®) on MG-63 (ATCC® CRL1427TM) human osteosarcoma cells at treatment depths ranging from 0.5” to 6.0”. The cells were grown to confluence on 4-well chamber slides that were kept in a nickel-alloy chamber within an incubator to shield the cells from unwanted environmental electromagnetic fields. During treatment, a specially designed apparatus held both the treatment device and the chamber slide. Briefly, the chamber slide was placed inside an acrylic tube at a specific distance from the transducer housing, and the device was turned on for 30 minutes. The chamber slides were then returned to the incubator to be evaluated at 7, 14, and 21 days post treatment for cell viability and bone nodule formation. Our results showed that compared to control cells, the cells located at 3” from the source had the greatest increase in bone nodule formation 7 days post treatment which is the depth at this consistent with manufacturer recommendations.

Evaluation of sperm motility with CASA-Mot: which factors may influence our measurements?

Computer-aided sperm analysis (CASA) is now routinely used in IVF clinics, animal breeding centres and research laboratories. Although CASA provides a more objective way to evaluate sperm parameters, a significant number of factors can affect these measurements. This paper classifies these factors into four categories: (1) sample and slide (e.g. preincubation time, type of specimen and type of chamber slide); (2) microscope (e.g. light source and microscope stage); (3) hardware and software, including the settings of each system; and (4) user-related factors. We review the effects of the different factors in each category on the measurements made and emphasise the need to take measures to standardise evaluations. The take-home message of the present article is that there are several commercial and useful CASA systems, and all are appropriate for routine analysis. Non-commercial systems may also be good choices when the user needs to adapt the device to specific experimental conditions. In both cases (commercial and non-commercial), it is important that standard protocols are put in place for evaluation, as well as methods to validate the system.

Biofilm formation by Pseudomonas aeruginosa and disinfectant susceptibility of planktonic and biofilm cells

Epifluorescence microscopy (EFM) was used to study the biofilm formation of Pseudomonas aeruginosa after 6, 24, 30, 48, 54, 72, 78, and 96 h growth in a chamber slide system. For this purpose, the biofilm was stained with the Live/Dead BacLight, wherein live and dead cells were visualised based on the cell membrane integrity. With the use of EFM we described 8- of 9-stage biofilm characteristics after 78 h of growth, since the majority of microscopic fields were fully covered with attached cells. However, the 96-h growth resulted in the cell detachment and revealed 30% of dead cells of all those cells that remained on the surface. The susceptibility testing of planktonic and biofilm cells to two disinfectants, chlorine-based and quaternary ammonium compound-based, revealed that biofilm cells were more tolerant to a chlorine-based sanitiser than planktonic counterparts. P. aeruginosa was inhibited by lower concentrations of the quaternary ammonium compound-based sanitiser than the chlorine-based sanitiser, which on the other hand was more effective in cell inactivation, as both the MIC/MBC (inhibitory/bactericidal) measurement and the CFDA/PI (carboxyfluorescein diacetate/propidium iodide) staining indicated.

Abstract P177: The Myotrophoblast of the Rat Placenta Ex Vivo Study of Nitric Oxide Synthase Inhibition

In pregnancy spiral arteries, are invaded by endovascular trophoblasts (EVT) and remodeled. Previously, NOS, and of smooth muscle proteins expression in EVT, and endothelin-1 (ET1) ex-vivo contraction of the remodeled artery were demonstrated, mediated by ET1 receptors A and B (ETA, ETB) Placentas on gestational day 21, were dissected, spiral artery rings devoid of smooth muscle were fixed to a silicon-coated 8-well chamber slide in oxygenated solution. Rings cut surface area (CSA) was observed under laser scanning confocal microscope. Following baseline, L-NAME and 10- 5 M, ET-1 10-7 M were added to some chambers. In other wells, also with ETA antagonist at 10-6 M (BQ-123). CSA was measured using ImageJ software. L-NAME alone, reduced CSA by 2.5% p=0.002. Addition of ET-1 to L-NAME, reduced CSA area immediately, compared with a plateau at 60min by ET1 p=0.001 .L-NAME, followed by ET-1 and ETA antagonist, the isolated constrictive effect of ET-1 via ETB, 7.2%, was earlier and stronger n than via ETA, 6.1% p< 0.001 (figure). L-NAME + ET-1 causes contraction of the arterial ring via ETA and ETB, without the dilatory effect of NO. ET-1 alone shows an earlier, immediate CSA reduction, compared to that of ET-1 without L-NAME, achieved at 40-60 minutes. This is in accordance with the instantaneous NO effect through ETB, compared with the gradual ET-1 induced CSA reduction .To isolate the contracting effect via ETB, we added L-NAME + ETA+ ET-1. The ETB contraction is earlier and stronger than that via ETA. Thus, EVT of the rat remodeled spiral artery react to ET-1 like vascular smooth muscle of non-modified arteries: contraction via receptors A and B and relaxation via receptor B through NOS.

Lab-Tek Chamber Slide for EM Prep: New Protocols for in situ Ultrastructural Study of Monolayer Cultures

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

The use of Lab-Tek tissue culture Chamber/ Slides and Flaskette for processing cultured monolayers for electron microscopy

In situ embedding is a preferable means for the ultrastructural study of cultured cell monolayers. However, the major difficulty is the separation of the polymerized plastic resin from the supporting substrates. This abstract demonstrates a simple, fast procedure for using existing products for cell culture and in situ embedding of monolayers. Epoxy block surfaces up to 10 cm2 can be easily separated from an untreated glass slide and sectioned vertically or horizontally for EM studies.The Lab-Tek tissue culture Chamber/Slides or Flaskette are constructed so that 1 to 8 chamber compartments or a single flask is attached to a regular glass slide by gaskets. Different cell lines or procedures can be performed on the same chamber/slide. When the plastic chambers and gasket are removed, the glass slide can be coverslipped and filed for reference. We use these products routinely for cell cultures. When an EM study is needed, we fix the cell monolayers in situ within the chambers or flask, dehydrate with ethanol, infiltrate and embed in an epoxy resin.