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!

The Shape Of Water

Water is the most abundant molecule in cells, organism, and on earth and it is so vital to life due to a combination of its unique chemical and physical properties.

Let’s first take a closer look at the structure of this essential molecule; water is polar (meaning that it has small charges on the individual atoms in the molecule which let it bond to other water molecules). This is so important and influences all other properties of water. Because water is polar, it can interact with other substances (such as salt) to dissolve them. In our bodies, all the chemicals are dissolved in water in our cells (in the form of the cytoplasm): water allows us to exist at a cellular level.

 

The solid form of water (ice) demonstrates another cool property of water as ice is actually less dense than its liquid form. We probably all take this surprising physical property of water for granted, however, for all other substances, the opposite is true. Ice is less dense than water because when the liquid cools down and molecules lose energy, more hydrogen bonds form between the molecules- forcing the molecules into a more spread out lattice to accommodate for the maximum amount of hydrogen bonds.

This unique physical property ensures that, even when its really cold, bodies of water such as lakes and oceans freeze from top-down – insulating the water below the surface and protecting aquatic life. This special property of water might have prevent organisms from going extinct in the past.

Water is so important to life that it is no wonder that we are almost constantly told to remain hydrated, especially in the summer. Naturally, in order to transport water, the majority of us use plastic water bottles: they’re convenient, lightweight, and cheap. But their hidden cost comes in the form of the damage it does to our health and the health of the planet.

The fact that plastic poses a risk to the environment probably doesn’t come as a shock to you. Despite this, you would probably still be forced to use plastic bottles since there are virtually no other cheap, lightweight alternatives that can even hold a candle to plastic. Until now…

Introducing Ooho! A water bottle that is completely edible and 100% biodegradable. Developed by Skipping Rocks Lab and inspired by the natural membranes found in eggshells, these orbs of water are encased in a calcium alginate gel layer and formed through a fascinating process known as spherification which was developed, not by chemists, but by chefs.

The process of spherification was pioneered in the 1950s by Unilever and it involves the reaction of two common chemicals which can form a tough membrane on the outside of the water- essentially, the water became its own water bottle through polymerisation. The first ingredient of the edible ‘water bottle’ is Sodium Alginate, usually derived from seaweed. This is a long-chain carbohydrate that is soluble in water. In its original plant, it is used to store sugars created by photosynthesis. When Sodium Alginate is dissolved in water, these long-chain carbohydrates float around on their own but they don’t connect to each other. That’s because poking out from these long chains is a branch of carbon and oxygen atoms, which chemists call an anionic group which has a slight negative charge. The sodium ions are attracted to this, because they have a positive charge. But sodium is monovalent, meaning that it wants to bond to just one of these carbon and oxygen branches at a time.

However, if you add calcium ions to the solution, the structure changes. Calcium ions have two positive charges, so they want to bond onto two of these branches at a time, meaning that it can connect two alginate molecules together. So the calcium ions replace the sodium and create cross-linkages between the long-chain carbohydrates. When enough of these chains are connected together, this 3D matrix of connected alginate chains forms a semi-solid impermeable membrane that the water can’t pass through -creating a sphere that surrounds the water. The fully formed water sphere is made from nothing but water and the two chemicals!

As part of the session, we experimented with sodium alginate and calcium lactate to design our own edible ‘water bottles’. We first dissolved one gram of sodium alginate powder into 250 ml of water to create sodium alginate solution, Then we dissolved five grams of calcium lactate powder into 1250 ml of water to create a calcium lactate bath. We then gently set a tablespoon of the sodium alginate solution into the calcium lactate bath- allowing the spoon to become completely immersed before tipping it over to pour out the sodium alginate solution.

The main difficulty was estimating when to remove the water orbs from the calcium lactate bath; we came to the conclusion that 20 minutes is the minimum amount of time needed for the membrane to solidify properly whereas leaving the ‘water bottles’ submerged for an hour ensured that the membrane remained flexible but strong.

Avenues for further investigation would be to freeze the sodium alginate into spheres and then submerge the solid spheres into the calcium lactate bath to produce ‘water bottles’ with a more defined shape and to experiment with calcium chloride which is said to produce a stronger membrane. Overall, however, I feel the experiment was fairly successful and we were able to sample numerous ‘water bottles’!

This technique is causing a bit of a stir in manufacturing circles as it could provide a waste-free way to contain and transport water as the membrane is both edible and biodegradable. It is still a long way from replacing the water bottle on store shelves, however, as the gel membrane breaks down over time and isn’t as tough as plastic.

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This blog post is based on the course content I created for a series of sessions on the biochemistry of water as part of the Exploring Everyday (Bio)Chemistry Society which I run at The Tiffin Girls’ School. The sessions focus on connecting chemistry with everyday products and phenomena to encourage KS3 engagement with science and they have been a great success so far. More blog posts based on this society can be found here.

In The Lab: Synthesising Polymers

In one of the earlier sessions of Exploring Everyday (Bio)Chemistry Society, we discussed the properties of natural polymers (including spider silk). Our focus now moves on to artificial polymers (including rayon and nylon). During our experiment based sessions  of this term, we replicated one of the earliest methods of synthesising rayon (a semi-synthetic fibre designed 125 years ago by the English chemist, Charles Frederick Cross).

First, a solution of tetra-amine-copper(II) ions must be prepared; I measured out 10 grams of basic copper carbonate and, working under a fume hood, added 100 cm³ of 880 ammonia solution. Despite operating in one of the best-ventilated labs, the choking scent of ammonia wrapped its tendrils around my airways almost immediately- suffocating me. I can safely say that the outcome of this experiment was still definitely worth it!

 

 

 

 

 

 

[Carefully measuring out the copper carbonate and dissolving it in ammonia to form a solution of tetra-amine-copper ions]

Next, I added 1.5 grams of finely shredded cotton wool to the solution. The cotton wool provides the cellulose (a natural reactant) for the reaction- which is why rayon is only a semi-synthetic polymer.

 

When cotton is dissolved in this solution, the resulting mixture becomes surprisingly viscous (with an almost shower gel-like consistency).

This initially presented a slight problem in achieving complete dissolution- a problem which was solved with the help of an exciting device I’d never had the chance to use before: a magnetic stirrer.

A magnetic stirrer consists of a small bar magnet which is dropped into the solution and a device which creates a rotating magnetic field – forcing the bar magnet to rotate and, hence, stir the solution.

The resulting effect is that the solution appears to stir itself – much to the shock and delight of some of the younger attendees of the session!

 

 

 

The final step is to simply inject a thin stream of the deep blue solution under the surface of some sulfuric acid.  As the solution enters the acid, its pH drops swiftly and the complex compound is converted into insoluble rayon.

 

Although the rayon initially appears deep blue in colour, as the copper (II) ions diffuse out of the rayon (reacting with the sulfuric acid to form copper sulfate solution), the colour quickly fades away- leaving behind only the delicate, translucent fibres.