How We Can Heal Fractures Faster

An approach using HAp/CTS nanofibrous scaffolds

Where the cuboid fracture is located (left) and star player George Kittle (right).

Not too long ago, around early November, one of the best players in the NFL got injured. The all-pro tight end had fractured his cuboid bone, rendering him out for the rest of the 49ers season, destroying their chances of a playoff appearance.

Being a fan of the team, I was extremely devastated to hear the loss of George Kittle. But I was even more devastated when I heard he would be out for most of the rest of the season, and to put that in perspective, that’s about 6-8 weeks. I just couldn’t grasp why a fracture would take so long to heal, despite all of our medical advancements.

So, I decided to look into it, and especially on how to improve healing times so injuries like this wouldn’t be as bad. After weeks of research and thinking, it finally clicked.

The Problem

To begin, the problem itself is filled with issues, ranging from expensive care to inefficient treatment methods.

The most common treatment methods for fractures are reconstructive surgery, medical devices (casts, splints, etc.), and sometimes physical therapy. All these methods are really expensive, and not to mention once the procedure is over, the healing time is multiple weeks or even longer depending on the severity of the injury.

Surgery is the most common method to treat serious fractures in order to either connect the broken bones, or stabilize them in place. Once this step is done, it usually followed by placing a biological scaffold around the fractured area inside the body (more on this later on) and a cast outside the body.

Even with all these new medical devices and innovations (most recently being that scaffold), fracture healing times still take awhile and puts a hold on one’s daily routine.

My goal was to find a way to improve the healing time, making it so patients can heal faster and more efficiently. I also wanted to be able to design something that could be scaled to other injuries such as skin wounds, nerve damage, cardiac tissue death, etc.

Keep reading to find out what I did.

Biological Scaffolding

To sum it up — scaffolds are the masterpiece of bone tissue engineering. A bone scaffold is the 3D matrix that allows and stimulates the attachment and proliferation of osteoinducible cells on its surfaces.

Summary of the bone scaffold process.

These osteoinducible cells are cells with a property know as osteoconduction. This is the ability of bone-forming cells in the fractured area to move across a scaffold and slowly replace it with new bone over time. Osteoconductive materials (which is what scaffolds are usually made of ) serve to allow bone cells can attach, migrate, grow and/or divide.

Essentially the more osteoconductive you can make a scaffold the better it will perform in growing more bone cells, which means a faster healing time.

The 5 main properties of a bone scaffold is that they should:

• Be biocompatible — cells must adhere, function normally, and migrate onto the surface and eventually through the scaffold and begin to proliferate before laying down the next matrix
• Be biodegradable — Scaffolds are not to be permanent, so it must be biodegradable as to allow cells to produce their own extracellular matrix at a certain point. The by-products of the degradation should also be non-toxic and able to exit the body without interference with other organs
• Be mechanically strong — Ideally, the scaffold should have mechanical properties consistent with the anatomical site into which it is implanted in, and must be strong enough to allow surgical handling during implantation. They also need sufficient mechanical integrity to function from the time of implantation to the completion of the remodeling process
• Be architecturally correct — The architecture of scaffolds should have interconnected pore structures and high porosity to ensure cellular penetration and adequate diffusion of nutrients to cells within the construct and to the extracellular matrix formed by these cells
• Be easily manufactured — In order for a scaffold to be clinically and commercially viable, it should be cost effective and it should be possible to scale-up from making one at a time in a research lab to small batch production

If you can make a scaffold with a certain biomaterial, and still have all these properties maintain, then that’s considered successfully. But this is no easy task, as there are various materials that can be used to fabricate scaffold. The main groups of materials fall into: ceramics, synthetic polymers, and natural polymers.

Each of these individual biomaterial groups have their own advantages and disadvantages, so picking the right combination can easily ensure success. Not only this, but even if you have the right solution of materials, you need to make sure you fabricate it the right way.

Similar to the groups of biomaterials, there are various ways to fabricate scaffold, each with their own advantages and disadvantages. Some of the popular ones are: freeze-drying, gas foaming, electrospinning, stereolithography, and bioprinting.

Unfortunately, unlike with biomaterials, you can’t really mix two methods to make a scaffold. You can only choose one, which is why it is vital to choose the one that best suits the solution you made. However, you are able to manipulate the way you actually fabricate the solution, as that almost always alters the end product.

Overall, picking the right materials, preparation method, and fabrication technique will lead to a successful scaffold. This is why it is important to weigh all the factors before experimenting, as one slight alteration can drastically change the outcome.

The Proposed Solution

As I said before, every little aspect of scaffolding is vital to success. So for my proposed solution, I made sure that I could ensure the success of the scaffolds by weighing out different scenarios before I jumped into it.

As a quick summary of my solution, here are the main components:

• Materials — hydroxyapatite/HAp (ceramic), chitosan/CTS (natural polymer), and ultrahigh molecular weight polyethylene oxide/PEO (synthetic polymer)
• Preparation — Using a co-precipitation method, the HAp/CTS nanoparticles were formed in a solution. The PEO was then added to this solution
• Fabrication — The solution was then electrospun into thin fibers, which then were shaped into a fibrous scaffold with the HAp/CTS nanoparticles embedded into the fibers

Now that you have a basic idea of what the proposed solution was, let me explain to you in much greater detail what the actual process was.

Choosing Materials

Like I said before, I ended up suing an HAp/CTS nanocomposite to make my scaffold, but let me break it down even further and explain my thought process behind each.

Overview of what goes into choosing materials for bone scaffolds.

• Hydroxyapatite(HAp) — This is a ceramic biomaterial whose nanoparticles must be formed via a certain preparation method. It is a major component and an essential ingredient of normal bone as it makes up bone mineral. It is this material that gives bones their rigidity. By getting a lot of it in a scaffold, you can help the bone get stronger by providing it with the biomaterial before hand, while stimulating surrounding cells to heal
• Chitosan(CTS) — A natural polymer, this biomaterial has been long considered as one of the most attractive natural polymers for bone tissue engineering owing to its structural similarity to the glycosaminoglycan (play a crucial role in the cell signaling process, including regulation of cell growth, proliferation, promotion of cell adhesion, and wound repair) found in bone, biocompatibility, biodegradability and excellent mechanical properties.
• Ultrahigh molecular weight polyethylene oxide (UHMWPEO) — A synthetic polymer. The purpose of this is to help fiber form stronger and better. The solution is added after the HAp/CTS solution is finished.

If you take a look at the 5 main properties a scaffold should have, you could see that these materials should ideally cover all of them. The HAp/CTS is biodegradable (its natural), biocompatible (cells have these materials), mechanically strong (chitosan), architecturally right (the UHMWPEO makes it so), and easily produced (materials are pretty common to find).

So after realizing that this combination of materials could potentially be an option, I decided to find out what to do with it and actually make it into a scaffold.

Co-Precipitation

To reiterate, how you prepare the solution is a vital step in making a good scaffold. Previous studies have simply mixed the solutions and electrospun them without any preparation methods, so it usually results in nanocomposites with very limited or lacking of specific interactions between the organic and inorganic materials.

Also, homogeneous dispersion of nanoparticles within polymer matrices is difficult to attain due to the nanoparticle ‘clumping.’ Weak molecular interaction and poor dispersion gives rise to problems like compromised electrospinnability, reduced nanoparticles loading capacity, decreased mechanical properties of the resultant nanocomposite nanofibers as well as unfavorable cellular responsiveness.

Enter the co-precipitation method. The purpose of this is to battle the issues aforementioned, and provide a way to produced the nanoparticles beforehand and then convert them into nanofibers, rather than just using solutions with various particles already there.

Here’s how the method works:

Diagram of co-precipitation method.

• Mixing Solution — Start off with mixing the two solutions together which then form into the HAp nanoparticles. In this case the solutions are a CTS/H3PO4 solution mixed with a Ca(OH)2 solution.
• Nucleation and Growth — After the solutions have been added, vigorous stirring occurs, followed by a period of ripening. This is when a crystal forms from the solution, in which a small number of ions, atoms, or molecules become arranged in a pattern characteristic of a crystalline solid, forming a site upon which additional particles are deposited as the crystal grows.
• Agglomeration — The sites made by nucleation, are where the HAp nanoparticles come and group themselves at these sites to form larger crystal HAp particles
• Precipitation — No, not rain. A precipitate is a substance separated from the solution usually as an insoluble amorphous or, in our case, a crystalline solid. The precipitate, containing the HAp nanoparticles, is separated from the solution
• Filtration — Pretty self explanatory, but essentially the precipitate is washed with deionized water for several times to neutral pH levels
• Calcination — The last step is when you heat the precipitate to high temperatures in air for the purpose of removing impurities or volatile substances.

Once the dried HAp/CTS nanocomposite has been formed completely, it is stored, usually in a vacuum oven, for subsequent use. At this point, the material we need for electrospinning is ready, so now we can prep it with the UHMWPEO and then electrospin the fibers.

Electrospinning

A novel method for fabricating methods as electrospinning allows for fibers with really high tensile strength. Here’s how it works:

Diagram of how electrospinning works along with the main components.

• As a simple overview, electrospinning is a technique in which charging liquid under high voltage leads to the interaction between the surface tension and electrostatic repulsion. This causes droplets on the spinneret needle to erupt and stretch
• A standard system contains 4 main components: a spinner with a syringe pump, a metallic needle(spinneret), a high-voltage power supply, and a ground collector (see above image)
• The strength of the electric field exceeds the surface tension of the droplet to produce a liquid jet that is then extended and whipped continuously by electrostatic repulsion until it is deposited on the grounded collector
• The solvent evaporates in the process, and the jet is solidified to form into a fibrous membrane

So for this solution, the prepared HAp/CTS nanocomposites are dissolved in a mixed solvent, along with the UHMWPEO, which will allow for fibers to form stronger, and easier. This solvent is then put in the syringe and then the electrospinning chamber.

The fibers are spun over a long period of time, until the desired amount has been formed. It is then vacuumed for over 1 week in a vacuum oven to remove any potential residual solvents. At this point a thin layer of solidified material of woven fibers should have been formed, ready to be shapen into the desired scaffold shape (ie. cylinder, cone, etc.).

So…Does it Work?

So, I walked you through the process of how to make the scaffold itself, but now only one question remains: how successful was it? I would say pretty good. There were a few issues that were resolved, but there are potential solutions to it.

Also, I have not actually made this myself as I don’t have access to the resources to do so. These results have been pulled from this research paper, which did a similar experiment as the proposed solution, so the results will be nearly identical. I have also talked to an expert in this field to gain some insight on this solution.

Here’s what the fibers turned out to look like:

The Good

Here’s what worked out:

• The conductive effect of the incorporated HAp nanoparticles stimulated a more significant level of bone cell formation ability when using the nanocomposite nanofibers of HAp/CTS scaffolds, owing to primarily the intrinsic osteoconductivity of HAp. The osteoconductivity has fundamentally been understood that HAp can absorb more proteins such as fibronectin and vitronectin, which will promote better binding with integrins (receptors that bind to the extracellular matrix).
• In addition to cell proliferation, mineral deposits were found to be much higher on the HAp/CTS fibers as compared to normal cell growth (ie. no scaffold). As higher amount of mineral deposits implies a higher degree of differentiation of the cells, enhanced bone formation ability can therefore be expected from the electrospun HAp/CTS scaffolds.

From these two major results, along with the fact that the scaffold didn’t collapse nor did it harm the cells, implies that overall the solution does work out. The HAp nanoparticles proved to allow more proteins to be absorbed by the bone cells, allowing faster regeneration and promoting ECM regeneration. Furthermore, the excessive mineral deposits on the fibers allow for stronger bone formation as unspecialized cells can quickly become specialized bone cells.

The Bad

Overall, there was only one major problem, but there might be a way around it. At the early stages of cell growth, the HAp/CTS scaffold showed significant low cell proliferation relative to the no scaffold. This is most likely related to the presence of ultrahigh molecular weight PEO on the fiber surfaces and/or in the medium, which could cause reduced protein adsorption and consequently delayed cell attachment and proliferation, which has been seen in other studies.

To alleviate this problem, a prior PEO extraction or leaching treatment is needed before placing it in with the cells. However, since PEO is water soluble and able to be released into the medium and gradually excluded during the cell culturing process, significantly high level of increase in cell proliferation after early days occurred and the scaffold composition induced differences in cell growth emerged.

After the initial inhibition period, HAp/CTS nanofibrous scaffolds had remarkably favored cell growth. By days 10 and 15, cell proliferation on the electrospun HAp/CTS scaffolds increased by approximately 43% and 110%, respectively.

Though the PEO did inhibit the cell growth in the early days, it did correct itself later on, and allowed for much faster cell growth. However, if we truly want a faster method to regenerate bones, then having faster cell growth in the early days is crucial. The only way to combat this is to remove the PEO once the scaffold has been completed. It is important to choose a method in which the HAp/CTS fibers won't be altered/destroyed.

This is something I hope to be able to test out soon, but due to my lack of resources and a lab, I am unable to do so. But from looking at the results and the solution overall, it seems like a worthy prospect for faster bone regeneration, despite the issues that come along with it.

Conclusion

To sum it up — in theory it should work. Without proper resources to test the methods due to COVID-19, there are some holes in the solution. But using analytical thinking, I have come to the conclusion that if the PEO was eliminated using a safe method (ie. not damaging the fibers), then the scaffold will perform much better in the early days, or will just keep the same properties as with the PEO.

I believe this is the next step in healing bones faster — enhancing scaffolds with nanotech, whether it be altering chemical properties, or structural properties, nanotech is the way to go.

I have thus far been researching in the field, and over a span of 1.5 months have come up with this proposed solution to battle the problem of slow, inefficient bone regeneration and do indeed believe it can change the world.

Hopefully, you got something out of reading this, and feel free to let me know if you want to learn more, or even you want to test this out at your own lab/have the resources to test this.

Key Points

To quickly summarize my thoughts, here’s a summary of the article:

• Bone regeneration methods are slow and inefficient. They can be improved via nanotech and biological scaffolding
• For a scaffold to be successful it must have 5 main properties — be biocompatible, biodegradable, mechanically strong, architecturally correct, and easily manufactured
• These scaffolds can be made by combining various materials, the major groups being ceramics, natural polymers, and synthetic polymers
• The proposed solution uses a hydroxyapatite/chitosan nanoparticle mixture as the solution, which is then turned into fibers, which is then shaped into the scaffold
• The HAp/CTS nanocomposite is prepared using a co-precipitation method which allows for better dispersion of the particles, along with stronger groups of them
• The solution is then prepared with PEO (a fiber-forming additive) and then electrospun into thin fibers over a long period of time to create a huge sheet of them
• The fiber sheet can then be used to shape the desired scaffold size and implanted into the body
• Overall the results prove that the idea would work, however there is one major issue — in the early stages the PEO inhibits the cell growth. To battle this an extraction of the PEO after the fiber sheet has been formed would be required

Thanks for reading, and as always, if you have any questions, comments, or opportunities, feel free to contact me at akashapatel2006@gmail.com or message me through my LinkedIn.

See you in the next one!