Tissue Engineering - Medical Engineering

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Microtubule Based Movement

V F Murphy Monday 19th April 2004

Cellular Motility is the movement of a cell, or the movement of environment past a cell. In simple unicellular organisms, the movement of cells through their environment is generally the focus, however, for larger more complex, multicellular organisms, the movement of the environment past the cell is more prevalent, such as the beating of cillia within the lung. Cellular Motility can be movement of a cell through its environment, or the movement of environment through (or past) a cell:Becker et al

Whilst there are a number of methods with which a cell may generate force, we will concentrate on force generation which involves the use of microtubles.

Kinesin and Dynein lie at the heart of Microtubule-Based Movement . These are termed Microtubule Associated Proteins (MAPs). These motor MAPs attach both to intracellular components, and to microtubles (MTs), and by moving along the MT they are able to transport the intracellular components, which could be organelles, or vesicles,…

How Kinesin Moves

V F Murphy Monday 19th April 2004

Kinesins typically consist of two large globular heads that allow attachment to microtubules, a central coiled region, and a region termed light-chain, which connects the kinesin to the intracellular component to be moved.

A polystyrine bead / kinesin / ATP combination was used to evaluate the velocity of kinesin anterograde transport (8nm per step, 2 micrometers per second), and both glass fibre and optical techniques were used to evaluate the force associated with this ( 8 pN forward), see references 1,2.

The movement described as walking involves one of the two globular heads moving forward, and making an attachment with a beta-tubulin molecule. As this bond is made, ATP is hydrolyzed, giving energy required to break the bond associated with the other globular head. In this way the molecule can process for distances as far as 1 micrometer.

The process of kinesin movement is ATP dependant, and is about 60-70% efficient.

Kinesin is in fact a family of proteins,…

Autonomous Organelles vs Centrioles

V F Murphy Thursday 15th April 2004

Plastids and Mitochondria

Both Plastids and Mitochondria are belived to result from primitive prokaryotic cells - this is termed the endosymbiont theory . An example is the Chloroplast which is believed to have evolved from the Cyanobacteria.

Evidence for this includes the fact that these contain their own DNA and the mechanisms to transcribe proteins - despite the fact that the vast majority of proteins within these organelles are also contained within the cell\'s nuclear DNA.

Plastids and Mitochondria are classed as semi-autonomous organelles because although they are capable of synthesis of their own proteins and contain their own DNA, within the cell they are partially reliant upon cellular metabolism in a way that cyanobacteria, for example, are not.

Centrioles

Centrioles form Centrosomes, which are nucleation points for microtubule organisation centres (MTOCs), which promotes polymerisation of alpha- and beta- tubulin dimers such that microtubles are …

Guide to Cell Counting

V F Murphy Tuesday 5th August 2003

So, you've a lot of cells and you want to know just how many? Cell counting is a key skill and it is crucial to use the correct proceedures.

In this tutorial we assume that you have obtained your cells in a suspension within an unknown volume of some fluid. If this is not the case, it is advantageous if the cells are manipulated to this stage before commencing counting. Resources Needed Hemacytometer - including cover slip

Microscope - many types are suitable

Handheld counter - of the click per count type, useful, but not essential

Gilson pipette - about 200uL

Tip for gilson pipette - 1 sterile, 1 not

Trypan Blue - small quantity required

Preparing sample It is essential that the cells are in a single cell suspension. Drawing up and expressing the suspension through a 23G needle and suitably sized syringe will generally achieve this. If the cells when viewed under the microscope still appear to be in clumps, then an incubation in pronase 10mg/mL for half an hour sho…

Hemacytometer Calculator

V F Murphy Sunday 27th July 2003

Our cell counting calculator simplifies the use of a hemacytometer - reducing the possibility of making potentially costly mistakes in your own calculations. It includes a guide to the use of a hemacytometer for cell counting. …

Collagen Sealant for Wounds

V F Murphy Sunday 27th July 2003

A team from Texas Agricultural Research Station reported a novel gel incorporating collagen for wound healing. The gel combines collagen with other materials, and speeds the closure of wounds, according to Dr Douglas Miller, one of the scientists working on the project.

Trials in rats showed comparable healing between untreated and treated wounds where injury ages were 6 and 3 days respectively, which the BBC expresses as a 50% improvement in wound closure. a 50% improvement in wound closure:BBC Benefits and Application Miller highlights the benefits of collagen, being an abundant protein within the body, so rarely producing allergic reactions. It is hoped that a major application of this wound healing product will be in diabetic ulceration.

The key to the new gel is that it is flexible and does not tear away from the wound surface as the subject moves during normal physiological activities. Miller hopes that future trials will take place, which may show a decreased visib…

Aseptic Technique for Tissue Culture

John Jones Monday 16th June 2003

The use of aseptic technique is essential for avoiding the production of infection whilst undertaking tissue culture activities.

Many activities take place within a laminar flow hood, either Class I (use with animal tissues), or Class II (use with human tissues).

The purpose of these hoods is to minimise the risk of infection entering from the outside environment, as well as protect the user against potential pathogens being transmitted from the culture into their environment.

A clean hood Before using a hood it is important that it is cleaned. This is typically by means of a chemical such as Industrial Methylated Spirits (IMS).

Cleaning the hood by means of swabbing with IMS or other suitable chemicals is very important. All items entering the hood from that moment on should also be treated with IMS to ensure sterility. An uncluttered workspace Reaching over items has a chance of knocking things over, or transfering infection through the workplace. For this rea…

Tissue Engineered Artery Progress

Based on the BBC Tuesday 10th June 2003

New blood vessels grown from a patient's own muscle cells are a step nearer after a genetic success in a US laboratory:BBC 06 Jun 03 The BBC reports upon a study undertaken at Duke University Medical Center in the US. Previous attempts at that center to construct tissue engineered arteries had fallen through based upon problems with limitations of SMC cell division. By introducing the hTERT gene into SMC Dr Laura Niklason and team were able to effectively immortalise the SMC, leading to the successful production of a structural artery.

Read the article in the BBC

Sources of Cells

James Anderson Thursday 5th June 2003

The source of cells to be used within studies are divided into two main groups depending upon the origin. The first group, Primary Culture, refers to those cells which are directly obtained from tissue, whereas Non-primary (Secondary, Tertiary, etc.) sourced cells are those obtained from existing isolated populations. Primary Culture Sources The primary sourced cells must be isolated from the tissue in some way. Most tissues surround cells with a biologically fabricated matrix which makes it hard to remove the cells. To effectively remove cells a number of techniques exist.

Isolation from fluid tissue

From fluid tissue such as blood the isolation of cells is a generally simple process. The cell populations may be differentiated by various centrifugation processes and more specialised techniques exist for hard to distinguish cells.

Solid Tissue - Outgrowth

First tissue is dissected into a number of fine pieces. These form the primary explants, and cells are encouraged to…