Bioreactor design, whether for research automation or scaling up of production, is a very common task for the Tissue Engineer today.  As an interdisciplinary field it is common to find people very skilled in one area, but lacking in another.  This article will shed light on a few things a mechanical engineer might think about when considering Tissue Engineering bioreactor design.

1. Disassembly:
   
   The golden rule is that everything should be easily disassembled and sterilised.  Do not glue parts together or fix them in a way that means there is (even potentially) an area which cannot be cleaned in the join.  Preferentially, mill parts from solid blocks or mould them in one part so that they do not need to be constructed from joined subcomponents.
2. Simplicity of Assembly:
   
   Don’t forget that your user will need to set up the system within a sterile environment, which often means a class II or higher tissue culture hood.  The user will be wearing rubber gloves and working at arms length whilst following the many restrictions of sterile procedure.  A good rule of thumb here is to design something that you could put together with one hand whilst sat on a chair arms-length away from the table you are assembling on.
   
3. Zones of sterility:
   
   Any material which contacts the culture medium or the cells must be both sterile and biocompatible.  It must not influence the culture or the cells by ions it releases.  A good option here is the use of a silicone or a perspex/acrylic part.  These can be sterilised with alcohol but do not last that many sterilisation cycles.  For short term use systems, stainless steel of grade 316 or 316L is possible; however these still release ions and cause problems for the cells in the longer term.  Any other non-contacting surfaces just need to be sterile – which means that stainless steel is a good choice, as is aluminimum where weight is a concern.  Make sure that the metal parts can be removed and autoclaved, and the plastic parts can be alcohol sterilised.
4. Operating conditions:
   
   Cells require definiate culture conditions, and these are usually provided in a laboratory through the use of a cell culture incubator in conjunction with a buffered medium that maintains the right pH.  This is a very good system, and you need a very good reason not to make use of it.  Preferentially, design your bioreactor to fit within a cell culture incubator – so that it can take advantage of the carbon di-oxide and temperature control.  To use a buffered cell culture medium you need to permit gaseous exchange between the medium and the atmosphere within the incubator so that the enhanced carbon dioxide level can be used for buffering – this must be a filtered gaseous exchange as the internal atmosphere of the incubator is not entirely sterile.
5. Heat and Humidity:
   
   An incubator or other cell culture environment will run at almost 100% humidity and operates at 37 degrees, conditions which mean careful device design.  In particular, electric motors will frequently overheat during use; or at the least, the heat they produce will overpower the temperture control.  A water cooling system is a good solution – this can be pumped or gravity fed (for short term experiments) from a reservoir outside the incububator, such that cool fluid runs through fine pipes over the surface of motors or other components which get hot.  Another very effective means of avoiding overheating and the negative effects of high humidity on electronics is to move them outside the incubator and make use of power transmission.  A wire-pull system will reliably move linear motion into a system, as will pneumatic or hydraulic drive devices.  These may have the added advantage of easier moving of the system from the tissue culture hood where it is assembled to the incubator where it will operate.
   

Structure, distribution, function

The lymphatic system forms a one way flow system towards the heart. Through this system flows lymph, which starts from blind ended capillaries. The capillaries are very permeable, and feature a loose overlap feature, where with the aid of small anchoring filaments the vessel can take large particles and stay open where the external pressure is greater. This causes problems however, as the vessel also carries large particles, such as viruses, pathogens, and cell debris, hence can carry infection through the body.

The Lymphatic System Diagram

Mechanism of transport

The capillaries soon aquire small one way valves, which ensure flow is only in one direction. The fluid is further moved through the system by means of compression caused by general skeletal muscle movements, including pulmonary inspiration.

Composition of lymphoid tissue

Lymphoid tissue is a whiteish tissue containing antibodies and immune cells.

Basic structure and cell population.

Major lymphoid organs:
Lymphatic capillaries are 10-50 micrometres in diameter, and are refered to as the initial (or terminal) lymphatics. They start from a blind sac, or from anastomising vessels. The endothelium is a single layer, with an incomplete basement membrane. There is a gap junction of 14nm, which makes it highly permeable to plasma proteins and carbon particles. The cell junctions are oblique, forming the flap valves noted above….the vessel also carries large particles, such as viruses, pathogens, and cell debris, hence can carry infection through the body:V F MurphyAnchoring filaments have been observed which keep the capillaries open at revers pressure gradients from the surrounding interstitium. The cappillaries then lead into collecting vessels, which flow into the afferent lymph trunks. Semi lunar valves in these larger vessels direct flow centrally, and the vessel gains a Smooth Muscle Cell layer and connective tissue.

Function

There are a number of other lymphatic organs which form semi-independant parts in relation to the main system, but the lymphatic circulation itself leads from a microcapillary filtrate from interstitium throughout the body, back into the blood at the L and R subclavian veins. Without this recirculation the cardiovascular system would stop in time, and massive swelling (oedema) would take place. The lymphatic system can deal with a 3 L / 24 hour imbalance of filtration of blood to tissue.

Lymphoid Tissues and Organs

The lymphoid tissues and organs house phagocytic cells and lymphocitic cells which are essential for the bodis defence against disease. The thymus and the spleen are other organs. Lymph nodes are integral tot he system and one single Lilum takes several collecting vessels into a bean shaped node. The node releases lymphocytes, and gamma globular antibodies. There is another unique circulation which provides nutrient to the nodes in the form of high endothelium venules.

While prosthetics is essentially a scientific field dealing primarily with helping amputees regain their ease of movement and ability, there is much research and interest in dealing with the psychological effects of amputation. From cutting-edge research in cognitive patterns of amputees to help quicken the rehabilitation process to helping patients deal with residual issues such as the constant realization that they’ve permanently lost a part of themselves, prosthetics is as much a science as it is about healing the minds of patients.

Phantom Pain originates not in the nerves but in the brain

Phantom pain is just one of such problems facing doctors and patients. Essentially, phantom pain is the sensation of a limb when it’s not present, or the extreme deformation or shortening of the limb (for example, some amputees have reported that they’ve felt their hands attached directly to their shoulders), sensation known as ‘telescoping’. Phantom limb pain is usually a burning, stinging, electric pain, and can increase under anxiety and stress. While it is quite common at initial stages, in time our body (and our nervous system) learns to adapt and the phantom pain tends to diminish and later on disappear (although it may reappear during periods of extreme stress or traumatic dreams). If it lasts, this may indicate a problem with the rehabilitation process or even a defect in the attached prosthetic. Then again, it could also be a case of our mind refusing to come to terms with the loss of a limb.

There are two main theories explaining phantom pain and they are both applicable to patients. The first theory contends that phantom limbs and phantom pain are mere expressions of the trauma of losing a body part experienced by the patient. Therefore, phantom pain is all in the ‘mind’, can be controlled, and perhaps eliminated. The second theory shows through empirical evidence that phantom pain is a direct result of nerve sensations (ranging from mild tingling to extreme pain). Within the medical profession there are conflicting views on the actual source of these sensations. One view is that phantom pain results as a consequence of the remaining nerves that keep generating signals. Another view is that in the absence of any (expected) response or sensory input from the limb, the spinal cord initiates excessive spontaneous impulses.

Although the first theory may seem simplistic in the face of medical evidence that clearly points to a mixture of excessive signals sent by the spinal cord and impulses generated by residual nerves, there is some truth in it. We, has human beings, are creatures of habit. Our bodies too have been attuned to certain types of sensory input and required responses. In the absence of such input (due to a missing body part) our consciousness (along with our body) takes considerable time to adapt. We might reach out for a glass on the table without thinking, and without the requisite limb, we may still get the feeling of an arm reaching out and a hand holding the glass. Such sensations are akin to ghost traces left in the cognitive and memory regions of our brains, and subsequently, our minds.

Running injuries and what the biomechanics of the human body tells us to prevent them

Running is without doubt the most popular of cardiovascular exercises. It forms an integral part of any serious fitness regime, and millions partake in the activity every year, be it for sport, training or just losing weight.

And yet, there is a serious downside to running most of us are not aware of. While it provides us with a variety of health-related benefits, studies show that around 60 percent of all runners experience some sort of lower-extremity injury due to the activity. The critical areas usually affected are your knees and ankles. Most of the running injuries are due to overuse, which occurs when your muscles, bones and joints are repeatedly subjected to high stress. The cumulative effect of these stresses can cause structural damage at a greater rate than at which the body can repair itself.

But running is not the actual cause of such problems. In fact, if you’re free from any joint-trouble and arthritis, and have not just come off the operation table with a serious operation to knee or ankle, the only factor causing just injuries is your running form. Many of us do not realize that not only do we need proper footwear and a suitable running track (hard pavement just wont do, it subjects your lower body to far greater stress), but we also need proper running form.

When analyzing how a person runs, we essentially look at their biomechanics and observe how optimal their ‘motion’ is. In other words, you’re looking at both the mechanical aspects of human motion and the efficiency of a person’s running technique. If a runner can minimize the stress to their lower body induced through regular running by simply maintaining proper form and technique (and obeying certain biomechanical principles), injuries resulting from running become rare.

The study of biomechanics deals with three important aspects: safety (freedom from injury), effectiveness (optimal level of performance) and efficiency (minimal effort for maximum output). When applied to running, most runners (especially if you run for sport), concentrate on effectiveness and efficiency. A thorough understanding of how the human body functions during motion can help trainers devise methods and programs through which runners can improve their performances (effectiveness) while conserving their strength (efficiency).

But what about safety? Everyone knows that you need to stretch before you run, to reduce the chances of cramps and poor running form later on. Most of us also try to adhere to proper footwear (running shoes instead of basketball shoes), and stick to designated running tracks where possible. However, the question of running technique is often overlooked and ignored. This has much to do with how we learn to run. Since that happens at an early age, little thought is put into the biomechanics of the activity. But how you run is critically important in the way that stress is distributed throughout the body. Simple things such as how your feet hit the ground and how your knees should bend during that time are major factors in determining how much stress is put on your body.

For example, roughly 80 percent of all runners strike the ground heel first. In heel-strike running, two problems occur. First, the leg is fairly stiff at the point of impact, resulting in a high level of shock to your knees. Second, heel-strike running results in extra pressure being put on the ankle. The result is heightened stress to your body, and a greater chance of injury through repetitive movement.

Instead, one should strike the ground with the midfoot (a practice supported by the majority of elite runners). This requires that you flex your leg upon impact, thus reducing the direct stress to your knees, as well as avoiding the extra pressure that heel-strike puts on your ankles. However, this is effectively a totally new method of running for our body, and one has to ‘unlearn’ basic motor patterns that haven been established since childhood. The benefits, though, are obvious. A greatly reduced risk of injury is a building block for serious running, be it for sport or fitness. Combined with proper motion techniques learned from a biomechanical analysis, you can easily learn to run safer, faster, and better.

Atherosclerosis is a disease characterised by the build up of scleroid lesions, termed atheroma. These atheroma take the form of porridgelike blockages within the human circulatory system.

These blockages act to distrupt the normal course of circulation, leading to infarction, whereby blood is unable to reach a certain area, or the formation of thrombosis, where the blood effectively clots within arteries or veins. These can lead to heart attacks, or strokes.

Assessment of the Arm Brachial Index, whereby the blood pressure ratios within the body are measured can point towards some form of circulatory obstruction, typically atherosclerosis. This test may be performed easily within a primary health clinic as a screening factor for such heart disease

If obstruction is suspected in a given area, it is possible to implement a doppler technique, which, through reflecting soundwaves from the region, is able to determine whether the flow is turbulent (mucky sound), or laminar (smooth sounding).

A more specialised technique is that of Ultrasonic Scanning, in which a high resolution ultrasound scanner is able to visualise obstructions, and possibly flow patterns surrounding the obstruction.

CT and MRI (MRI arteriography, MRA) can both be used, however, this tends to be for presurgical inspection, rather than initial diagnosis.

The main point to take away from this review is that it is generally impossible to diagnose atherosclerosis until it is suspected, for example, following minor heart complaints, difficulty walking, or similar. Routine assessment for at risk groups has been implemented within some countries.

Structure of Muscle

Muscle is made up of fibrous components termed myofibrils. Each of these is formed by a large number of sarcomeres. Each sarcomere feature repeated bands of Actin and Myosin, causing a banded apperance during microscopy.

The Sliding Filament Model

Huxley proposed that not only did muscles contract by changing the degree of overlap between the actin and myosin filaments, but the force was determined by the degree of such overlap.

Testing the Sliding Filament Model

Microtubles are key to the movement of cells, especially cellular appendages. Cillia and Flagella are the principle appendages concerned with such motion.

Cillia are intracellular structures, as they are within the plasma membrane, however, they are extrusions from the shape of the cell into the surrounding environment. Cillia generally are grouped on the surface of cells, termed Cilliated Cells.

Cilia move in a coordinated way, and with a single motion described as \’oar like\’. The cycle takes about 0.1-0.2 seconds to complete.

Flagella (plural of flagellum) are designed more for cell locomotion, and are also termed intracellular. In most cases these are located behind the cell itself. Beating is by sequential undulations, leading to the propulsion of the cell through a fluid environment.

Structurally, both Cilia and Flagella consist of an Axoneme connected to a Basal Body.

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, to where they are required. In this way, MAPs can be visualised as trains running along MT tracks.

Motility is driven by ATP (Adenosine-Tri-Phosphate), the hydrolysis of which acts through specific proteins to mediate movement. Dynein has been differentiated from kinesin based upon its direction of movement – kinesin moves away from the negative end of a MT, i.e. away from the MTOC or centrosome, and towards the distal portion, termed retrograde axonal transport. Dynein however moves in the opposite direction, acting to move items closer to the MTOC, termed anterograde axonal transport.Dyein Decends toward the MTOC, wherease Kinesin Klimbs away from the MTOC:MedicalEngineer.co.uk

In the case of neurons, clearly defined axons are characterised by bundles of MTs. Here it can be demonstrated that the combination of kinesine and dynein act to move proteins to the end of the axons, the synaptic knobs. In the case of the squid giant axon, this distance can be up to a meter – allowing for proteins to reach areas far from possible protein synthesis apparatus. This is known as fast axonal transport – a mechanism very similar to that which is generally accepted to be displayed within other cells. It is possible for organelles to move at a rate of approximately 2 micrometers per second.

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, including some which move towards the negative end of the MT. These different Kinesins act for different purposes, and have different structures, including mitosis and meiosis.

References

1.
Proc Natl Acad Sci U S A. 2003 Mar 4;100(5):2351-6. Epub 2003 Feb 18 : Probing the kinesin reaction cycle with a 2D optical force clamp : Block SM, Asbury CL, Shaevitz JW, Lang MJ.

2.
Biophys J. 1996 Dec;71(6):3467-76 : Kinesin force generation measured using a centrifuge microscope sperm-gliding motility assay : Hall K, Cole D, Yeh Y, Baskin RJ.

3.
Benjamin Cummings 2003 : The World of The Cell : Becker, Kleinsmith, Harding

Cells, especially eukaryotic cells, have prominent delimiting thin films which are termed \’cell membranes\’. These membranes implement a vast array of cellular functionality, from retention and seperation, to the transport of solutes.

The primary roles of the cell membrane are to:
- Form the boundary of a cell
- Provide a reaction and attachment surface
- Permit transport of substances in and out of the cell
- Hold equipment that senses extracellular parameters
- Permit cell-to-cell signalling (intercellular signalling).

The structure of cell membranes was discovered by Signer and Nicolson to be that of the \’Fluid Mosaic Model\’. This model is generally accepted as the membrane structure. In this article, we discuss only this Fluid Mosaic Model, as it is universally recognised as accurate.

The Fluid phase of the membrane is made up of Membrane Lipids. These make up a lipid bilayer, first proposed by Gorter and Grendel. The bilipid layer means that there are two layers of lipid, each opposing one another, with the hydrophobic \’heads\’ facing the inside and outside of the cell, they hydrophillic \’tails\’ making up the internal meeting points. These lipids include Phospholipds, Glycolipids, and Sterols.

These lipids are both fluid (i.e. they can move around the membrane), and asymetrically distributed between the two layers.