Give Me A Break: Bioengineering Solutions to Spinal Fracture

I’m writing this post in a lab at Queen Mary University in the heart of London. Through the EDT Headstart scheme, I have been given the brilliant opportunity to immerse myself in the field of bioengineering (from the physiological level right down to the molecular level) surrounded by leading academics and university lecturers as part of a 4 day residential course.

Absorbing fascinating new information about molecular bioengineering has been a major highlight of the course. On the first day, I attended Dr Karin Hing’s lecture ‘Engineering Biocompatibility for Bone Regeneration’ which inspired my independent group research project on the use of synthetic bone grafts, autografts, and growth factors in spinal fusion.

Biocompatibility lies at the intersection between biology and material science and it refers to the interactions between an artificial material and a living system. With its abilities to bear loads and control homeostasis as well as self-regulate and repair, bone is a particularly interesting natural material – but what is it actually made of? Bone primarily consists of inorganic materials, such as calcium hydroxyapatite, and organic materials, such as the protein collagen, which make up a rigid extracellular matrix. Within this matrix, resides a range of specialised bone cells: osteoblasts, osteoclasts, and osteocytes.

Like ourselves, bone has a life cycle. Our bones are constantly undergoing remodelling, cycling between phases of formation and resorption. In formation, mesenchymal stem cells in the bone marrow differentiate into osteoblasts (bone-forming cells). These osteoblasts create the extracellular matrix (known as the osteoid) and mineralise it with calcium deposits. Next, they find themselves trapped within the bone matrix and they mature into osteocytes which are no longer capable of bone formation, instead serving as a communication port- relaying signals between other bone cells (much like neurons).

Life is centred on balance, a yin and yang of sorts; as new bone is built up, old bone is destroyed through the process of resorption. Osteoclasts are a key player in resorption and prevent new bone formation from getting out of hand. Bone tissue contains rich deposits of minerals such as calcium and, when it is resorbed, the minerals leach out and enter the neighbouring blood stream. Through selective resorption of bone tissue, osteoclasts maintain homeostasis and regulate our calcium levels. Between formation and resorption is the reversal phase in which mononuclear cells inhibit osteoclasts and prepare the bone surface for osteoblasts to build the connective tissue up again.

With its constant repair and remodelling, bone can automatically fix virtually any hairline fracture or microdamage. But when it comes to breaks larger than 2.5 cm (known as critically large defects), the bone can no longer spontaneously heal and will require medical intervention. For instance, in cases of vertebrae fracture, spinal fusion is necessary to heal multiple vertebrae into a single, solid bone – restoring stability to the spine.

What do we use to bridge the gap between vertebrae? That’s where bone grafts come in. Traditionally, a bone graft involves removing a small portion of bone from the hip (where it will naturally heal back) and transplanting it between the vertebrae to promote bone growth at the fusion site. If the graft comes from the patient’s own body (which is currently the gold standard in spinal fusion) then it is referred to as an autograft. Allografts come from a cadaver or live donor and are used when the fracture is too large to bridge with the patient’s own bone.

harvesting an autograft from the iliac crest (hip)

The reason autografts are considered the gold standard is because they can integrate into the patient’s fracture more rapidly and completely and there is no risk of immune rejection or bio-incompatibility. However, autografts require the patient to undergo two surgeries – giving rise to an array of new complications including donor site morbidity and secondary infections. Additionally, multiple incision sites may be necessary to extract an ideal autograft which increases the risk of nerve injury and bone damage at the donor site. But allografts are not without their flaws either; they come with a risk of immune rejection and disease transmission and there are long waiting times to even obtain an allograft due to a shortage of eligible donors.

Recently, synthetic alternatives to bone grafts (such as calcium sulfate and collagen) have emerged. Initially, these synthetic bone grafts were bio-compatible but still did not promote bone healing. Let’s take a closer look into what is necessary for a graft to support successful bone formation. Firstly, we have osteoconduction which is the graft’s ability to support the attachment, migration, and growth of osteoblasts within the structure. Then, there is osteoinduction which refers to the graft’s ability to stimulate stem cell differentiation into bone cells (osteogenesis).

Ever tried gardening? The properties of an ideal bone graft are similar to those needed to grow a plant. Osteoconductivity (the scaffold properties of the graft) can be seen as the pot and soil, osteogenesis by the actual seeds, and osteoinductivity is best represented by the fertilisers and water that stimulate plant growth.

At the very least, a synthetic graft must be osteoconductive for a successful fusion. On the third day of the course, I had the chance to enhance my wet lab skills via testing protein adsorption in a range of synthetic grafts. The experiment involved incubating five types of graft material (both organic and inorganic) in an albumin solution and using a colorimeter and calibration curves to assess the efficiency of protein adsorption in each of the graft types.

The first synthetic grafts were bio-compatible but also bio-inert meaning that they had were osteoconductive but not osteoinductive. To overcome this issue, subsequent designs of synthetic grafts tend to be infused in growth factors. Growth factors stimulate differentiation of stem cells and growth of living tissue – accelerating bone formation.

The most widely studied growth factor is bone morphogenetic protein 2 (BMP) which acts as a chemotactic agent by recruiting stem cells to the fusion site and prooting their differentiation. BMP even stimulates the growth of blood vessels (angiogenesis) through the damaged bone which helps support new bone formation.

the structure of BMP-2

BMP is considered safe for use in spinal fusion and eliminates the need to take a large portion of the patient’s own bone cells in a graft. The only downside is that it is currently very expensive – increasing spinal fusion surgery costs by up to $15,000.

BMP works alongside, and can be incorporated into, both types of graft. Synthetic grafts can be made of bioabsorbable polymers (such as lab-grown collagen or calcium sulfate structures) which carry doses of growth factors. As the material dissolves in the body, the BMPs are gradually released, helping sustain bone growth. In autografts, the patient’s own cells can even be genetically engineered to express specific growth factors in greater quantities.

It has become increasingly clear that bone grafts cannot be developed solely from an engineering perspective. As Dr Alvaro Mata explained in his lecture on bioinspired materials, nature is a particularly important source of inspiration for designing any bioengineered system, particularly synthetic bone grafts. Structure at even the smallest levels, such as the arrangement of structural protein nanofibres, affects cell signalling and growth to a large degree.

Biomimetic design is developed through an appreciation of nature’s own design. Through dissecting a bovine metacarpal joint, I gained a far better understanding of the interplay between bone and cartilage that makes smooth movement effortless.

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As the end of my week at Queen Mary’s bioengineering department crept closer, my team began to prepare a presentation showcasing our independent research in the fascinating field of spinal fusion. This presentation was the perfect culmination of the amazing molecular bioengineering techniques I had studied and carried out in the lab. To my absolute delight, we were awarded the 1st place prize for our level of detailed research and our debate style of presentation – a rewarding end to an incredible week!

Ri Summer School: Forensics

Every summer I look forward to attending some of the several Summer School workshops organised by the Royal Institution; this year I particularly loved the LYSC Forensics workshop (01.08.18) which focused on the DNA fingerprinting process to identify and compare DNA samples.

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Understanding the basics of DNA can help us get a better understanding of forensics and how techniques can be used to identify criminals from only a few drops of blood at a crime scene.

DNA (deoxyribonucleic acid) is a long molecule which consists of billions of smaller units known as nucleotides. Just as there are differences in our appearances, there are also differences in our DNA which are known as genomic variations. The genomic variations between people tend to be very minor and the more closely related two people are, the smaller the variation between their genomes. Two random, non-related people would have 99.9% of their DNA in common and yet there are still more than three million differences between your genome and anyone else’s.

These variations arise due to mutations – changes that occasionally occur in a DNA sequence during replication. When a mutation occurs in a in a sperm cell or an egg cell, it will be passed along to the offspring. Your genome contains around 100 mutations which make it unique.

Most genome variations are relatively minor; to help put this into perspective, I like to visualise the human genome as a book. If you’ve ever been to the Wellcome Collection, you might have discovered the Library of the Human Genome – an expansive bookcase containing a human genome typed out in a series of 109 books.  With that in mind, the books of your genome and mine would essentially tell the same story but yours might have a typo on page 17 of book 56 whilst mine might be missing a few letters on the penultimate page of book 34.

(Credit: Wikipedia)

The differences seem so small that it is remarkable that they can be detected through the use of forensic techniques.

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The workshop explained and guided us through the restriction fragment length polymorphism (RFLP) technique of DNA fingerprinting; this process was the first DNA profiling technique which was inexpensive enough to see widespread application.

In the workshop, we were presented with (isolated) DNA found at a hypothetical crime scene and samples of DNA from five ‘suspects’. By adding a restriction endonuclease (a type of digestive enzyme produced by bacteria), we chemically ‘cut’ the DNA into fragments. My work experience at the Centre for Liver Research proved extremely helpful during the workshop as I was already comfortable with using a micropipette to transfer and mix substances.

The next step in the process involved separating the fragments through the process of gel electrophoresis. Gel electrophoresis takes advantage of the fact that DNA is negatively charged and that, due to genomic variation, the isolated fragments of DNA will be of different sizes.

First, we prepared the gel by pouring agarose into a casting tray and pressing a well comb into the molten agarose. When the agarose set, the comb was removed to create a series of wells in the gel.

The fragments are separated by passing an electric current through the gel. This causes the negatively-charged DNA to move from the wells to the positively-charged electrode through the agarose. Shorter fragments can move easily through the pores in the agarose and so will move faster and migrate farther than longer fragments in a given time. This separates the fragments of DNA into bands. The unique pattern of bands of the suspects’ DNA can then be compared to that of the DNA found at the crime scene.

(A blue loading dye was added to the DNA samples before pipetting them into the gel. The dye is co-migratory and so will separate and migrate at the same rate as the DNA fragments. )

After the gel was run, we used a transilluminator to better see the bands of DNA fragments.

From this, we could conclude that the DNA found at the crime scene belonged to Suspect 3 – the experiment was a success!

 

What’s in a baby’s kick?

I am delighted to have been able to attend a Royal Institution ‘Summer School’ workshop on bioengineering this August. The focus in this workshop was on refining drug delivery systems and using mathematics to determine whether a foetus is developing properly.

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The first half of the session was lead by Rachel Dorris, a medical physicist, and was centred on how drugs can be better designed for use in inhalers.

Asthma is an autoimmune disease caused by the spontaneous contracting of the smooth muscles that surround the bronchioles in the lungs which results in constricted airways. Inhalers contain a drug (a bronchodilator) that can ‘open up’ the airways.

 

(credit: slideplayer)

In order for this treatment to be effective, the drug uptake (the retention of the drug in the necessary organ) must be at its optimum. To measure the drug uptake, a medical imaging technique that suits this purpose must be chosen. After some collaboration, our team decided that a Nuclear Medicine Scan was the ideal technique. It involves giving the patient the bronchodilator with a small amount of a radioactive tracer (Technetium-99m) and then observing the patient with a gamma camera. The image quality is relatively low but provides functional information and can be easily used to observe drug uptake.

This is an example of a Nuclear Medicine Scan of the lungs. The whitest areas show the highest drug uptake. (credit: slideshare)

From Nuclear Medicine Scans of 4 different drug particle sizes in patients’ lungs, we performed a qualitative analysis of the images- determining which particle size we believed would have the optimum drug uptake in the bronchioles. We then performed a quantitative deposition analysis of the images to confirm our ideas. The conclusion we arrived at is that a bronchodilator with a particle size of 1.5μm was best suited to use in inhalers.

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The second half of the workshop was lead by Dr. Stefaan Verbruggen, who is currently researching the effects of kicking in the womb during pregnancy on the development of musculoskeletal diseases, and, in particular, developmental dysplasia of the hip (DDH).

The hip is a type of “ball-and-socket” joint. In a normal hip, the ball at the upper end of the femur fits firmly into its socket in the pelvis. In babies with DDH, this joint is not properly formed and so the ball does not fit well in the socket and is easily dislocated. In all cases of DDH, the socket (acetabulum) is shallow which means that the ball cannot fit inside it comfortably.

 

(Credit: Oxford University Hospitals)

1 in every 1000 babies are born with a form of DDH. There are varying degrees of severity in DDH cases:

• Subluxatable- the ball of joint is simply loose in the socket
• Dislocatable- the ball lies within the socket but can be dislocated easily
• Dislocated- the ball is completely out of the socket.

(Credit: helpmegrowutah.blogspot.co.uk)

DDH is not genetic, but the risk of a baby having DDH greatly increases if there was not enough room in the womb whilst its bones and joints were developing.

A foetus’ bones and muscles, like in adults, react to stress by growing. In adults and children, the main stressor acting on the hip joint is gravity, but this is not the case in foetuses. Instead, the main way that foetuses can ‘exercise’ their hip joints, is by kicking. Considered one of the most endearing interactions between a foetus and the outside world, kicking is vital to its development; if there is not enough room for a foetus to kick, complications with joint development and DDH can arise.

• It is a ‘breech baby’ (the foetus is positioned feet first). In scans, it is revealed that these babies cannot fully extend their legs when kicking.
• There are issues with oligohydramnios (in which there is less amniotic fluid- normally due to a fault in the foetus’ urine production so that it ingests the amniotic fluid but does not pass it out). Notice how, in scans, these babies don’t appear to kick at all.

Cine-MRI scans of baby kicks

 

Curiously enough, foetal twins tend to have enough space to kick comfortably, despite having to share the womb. (They also are unable to kick each other due to a membrane wall separating them!)

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The progress of a foetus’ joint growth and muscle activity is measured to ensure that the pregnancy is going well and to calculate the risk of DDH. However, when foetuses are in the womb, experimental equipment, naturally, cannot be used and only images can be used. This means that mathematics must be employed in a fascinating manner to calculate strength of a kick from only two MRI scans.

 

Start of kick (Frame 76)

End of kick (Frame 83)

First we must work out the change in angle from the start to the finish of the kick. At the start of the kick, the angle between the hip and the ankle is 87°. At the finish, it is 153°. This means that the change in angle is 66°. Then, this must be converted to radians, making the value 1.15192 radians.

The MRI scan was taken at 3 frames per second and the kick took 8 frames. Using this, we can calculate that the length of the kick in seconds is (to three decimal places) 2.667 seconds.

Speed is found by dividing the distance by time taken to complete the distance. In this case, the distance is the change in angle. Therefore, the speed (or velocity) of the baby’s kick is  1.15192 / 2.667 = 0.432 radians/s.

The length of the lower leg is 58.4 millimetres and it can be considered a radius in this case. Acceleration is found by multiplying the square of the velocity by the radius. So the acceleration of the kick was 0.432² x 58.4 = 10.899 mm/s²

The foetal leg would weigh 0.321 kg. Since force = mass x acceleration, we can now also find the force that the foetus must generate to perform this kick- 3.498 N.

This example demonstrates just how powerful mathematics is; using only 6 relatively simple calculations, we can quickly work out the force of a foetus’ kick from just 2 basic MRI scan frames.

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Overall, the workshop was an incredible introduction into the field of bioengineering. Without a doubt, the second half of the workshop was my favourite as I found the exploration of DDH in babies very interesting and I was amazed by how easy a seemingly impossible task was made by using formulae that I had already learnt in school but had never seen being used to solve ‘real-life’ problems.