How 3D printers might make you live longer
We should all envy Wall-E.
No, not because of his nice cricket pet or his boiled-egg-looking girlfriend who tries to shoot scorching lasers at him (sorry, Eve).
It’s because he remained functional and built mountains of cubed trash over a loooong period of time even after the humans left earth on a fancy space cruise!
How was he able to do so? Well, first of all, he was solar-powered 🌞, but most importantly, he could replace any broken parts for a better, functioning one (you might remember his Ferris wheel-inspired shelves 🎡 storing his extra eyeballs. Look here for context).
Unlike him, we humans die relatively early because we cannot fully repair broken body parts. Sadly, it’s a bit more complicated than flicking on and off wires 🔌.
Luckily, we might be able to make brand-new body parts customized just for each individual in the future! How? Tissue engineering.
Here, grab a seat. In this article, I’ll tell you all you need to know about tissue engineering, how it works, and what you should expect in the future.
The problem with organ transplantation
Tissues are like cars. When the tissue gets damaged, there are two options for treatment — fix it 🛠 or get a new one.
Currently, fixing your organ is cheaper and widely available, but when your tissue gets so damaged that it cannot fix itself, tissue replacement is the only choice 😢. Some tissues, like cardiac muscle tissues, don’t even have an effective repair system, to begin with — they’re poor little cars that auto repair shops don’t accept.
For right now, the only one way to get a new tissue is through organ transplantation from a human donor or an animal (called xenotransplantation, not commercially available), most commonly a pig 🐷.
Even as you’re reading this article, one person dies every hour waiting for an organ transplant and one person is added every ten minutes to the transplant waiting list. It creates a pretty morbid relationship, too — someone else dying is of benefit to people on the waiting list. And yes, this is why we have organ trafficking.
Even if you luckily receive a donor organ, there is no guarantee that it would properly function, or even survive in your body. In fact, the patient’s immune system might attack the new organ just as it would a cold virus 😱.
What is tissue engineering?
Tissue engineering is fabricating new tissues to replace damaged ones by arranging living cells into the shape of a tissue and letting the cells proliferate and develop into a working tissue.
The goal of tissue engineering is to create long-lasting tissues that fully reproduce the function of normal tissue. In other words, they could be integrated onto the host’s body and begin fulfilling its role in the body (ex. pumping blood for hearts) like a real tissue, except much better than the damaged tissue it’s replacing.
Have you ever ordered clothes online and realized they don’t fit? It sucks 😤. It would be even more terrible if you ordered your organ (assuming the tissue engineering technology is established) and it failed to properly work in your body 🤒.
Well, don’t worry.
Each engineered organ is customized to the physiological characteristics of each patient, like their height, weight, the shape of damaged tissue, etc, based on the MRI/CT image of their body (more on this later), so at least geometrically, the engineered organ are designed to fit each individual patient.
The engineered tissue can also be built using cells collected from the patient, meaning that the patient’s body would automatically recognize the engineered tissue as an original body part and not attack it.
Originally, tissues were made by pouring cell-containing material into molds and letting it cure. This method is hard to control and ineffective, so scientists now use 3D printing to fabricate tissue scaffold that predetermine which shape the tissue will grow into.
Pathway of Tissue Development
How a bioprinted tissue scaffold develops into a functional tissue that can perform biological functions is similar to how tissues form during the natural growth of an animal. Here’re the main cellular activities that guide tissue development 👇
- Cell proliferation. When our embryo develops into a complete human, the cells making up the embryo undergo extensive cellular division (aka cellular reproduction) to create replicas of themselves.
- Cell differentiation. Some of these replicas start activating a unique set of genes to have a super-specific function within an organ and become differentiated cells like muscle cells and red blood cells.
The undifferentiated rest persist throughout the organism’s development just for the sake of reproducing cells that are available to differentiate when some of the current differentiated cells die. These cells are called stem cells, like the stem of a plant 🌿 where multiple cell lineages (the leaves) can emerge from.
3. In conjunction with cell differentiation, the replicated cells migrate into specific places within the embryo to organize into tissues like how the brain cells would move to the top portion of the organism’s body, where the head would form.
4. To form working tissues, the cells excrete biological glue called the extracellular matrix (ECM).
The ECM fills up the space between cells and integrates them into the form of a tissue (without the ECM to tie the cells together into a stable sheet of co-living cells, the cells would simply be cornflake cereals floating around your body 🥣).
The ECM also acts as a medium through which cells exchange signals, nutrients, and molecules to each other, and regulates which of them get to enter the cell.
Besides the ECM, the cells also adhere to their neighboring cells to perform short-distance, direct communications. Think of these short-distance interactions like quick notes that you secretly pass around your friends during math class. Communication through the ECM’s, on the other hand, are letters you send through USPS to your friend in LA. (I’m not using a texting analogy here, because all communications in cells occur physically by molecules; sorry, no cell telepathy 🥺).
Overall, communication allows cells to coordinate their actions to best respond to the environment. Poor communication would result in something like a chaotic kindergarten class, where the kids don’t listen to the teacher (the ECM) and the class gets nothing done.
5. Lastly, blood vessels form in the tissues during angiogenesis. Blood vessels carry nutrients and oxygen to the tissues and take away cell waste, so that they don’t build up and harm the cells.
Particularly, a subset of blood vessels called capillaries infiltrate into tissues and branch off in all directions.
Layers of tissues, each tissue serving a role of its own, are organized into organs that perform larger functions in the body, like the heart to pump in/out blood. With capillaries supporting each tissue layer, an organ consists of multiple layers of interconnected blood vessels.
To recap, cells divide, differentiate, migrate, produce ECM, and create blood vessels in tissues during tissue development.
So how exactly do bioscaffolds guide cell behavior? Bioscaffolds act as artificial ECMs that guide the cells’ behavior during tissue development. The mechanical and properties of the scaffold’s surface come in direct contact with the cells, so these properties are precisely adjusted to signal the cells to act in certain ways that ultimately led to cells developing into the desired tissue.
Bioscaffolds have fabricated a technique called bioprinting, which is a type of 3D printing (3D printing: building a 3D structure from bottom to up based on a 3D image).
Inside the bioprinted scaffold, the cells proliferate to fill up the scaffold and secrete their own ECM that completely replaces the hydrogel material in the scaffold. The tissue is developed once the cells function as tissue and all the hydrogel material is replaced
It’s a bit like the balloon chocolate bowls, where you pour chocolate onto a balloon and pop the balloon after the chocolate hardens so that the chocolate keeps the shape of the balloon. The balloon is the scaffold material, and chocolate hardening is cells dividing and differentiating to make tissue. Even when all the scaffold material is degraded and removed, the resulting tissue remains in the shape established by the scaffold.
So, how do we make tissues?
Here’s a quick summary of how tissues are made by bioprinting scaffolds:
- Image generation. 3D imaging — CT or MRI — of the target tissue is reconstructed as a CAD model and translated into .gcode, which is read by the bioprinter.
- Cell culture. Produce the number of cells you need to embed in the scaffold (and I’m talking millions of cells here)
- Bioink making. Mix precise concentrations of materials — hydrogel, cells, crosslinkers — into a bioink, and load it in the bioprinter.
- Scaffold printing. Allow the bioprinter (a type of 3D printer) to construct the scaffold based on a digital 3D model of the tissue.
- Scaffold culture. Incubate the resulting scaffold in an environment that mimics the conditions in the internal body and supply the cells with nutrients to grow, differentiate, migrate, and produce their ECM.
- Implantation. Insert the functioning tissue into the patient’s body by surgery. After implantation, the new tissue would undergo angiogenesis with the patient’s body, where the patient’s blood vessels grow their way into the engineered tissue.
Image generation
At the heart of our excitement for tissue engineering lies the customization aspect of engineered organs. The bioprinter creates the tissue scaffold based on a 3D image of the patient’s body, either by a CT or MRI scan.
(So, precisely what part of the patient are we modeling? Is it the damaged tissue? The surrounding physiology? There hasn’t been a standardization of how 3D print images are customized to the patient, but several options include trying to model the healthier version of the damaged tissue or adjusting the dimensions of a standard patient heart model to match the size of the patient)
Computer tomography (CT)
A CT, or computer tomography scan, is created by shooting a beam of x-ray at the patient’s body, layer-by-layer in a helical pathway: the source of laser spins around and along the patient’s body to take multiple images in a shorter amount of time. You can watch how this helical imaging works here.
On the other side of the x-ray source is an x-ray detector that measures the amount of x ray that has been absorbed by the body ( →gives off information about 3D volume and composition of the body).
CT scans give us images of cross-sections of our body, and we can combine these cross-sectional layers into one 3D model of what our body looks like. Unlike normal x-ray scans, CT scans are much more detailed than x-ray. and shows soft tissues, bones, and organs.
Magnetic resonance imaging (MRI)
Magnetic resonance imaging uses a very powerful ring-shaped magnet that detects the magnetic field created by protons to make a 3D image of the human body.
About 60% of the human body is made up of water. The nucleus of hydrogen atom in water and fat molecules house positively charged particles called protons.
As a review for physics, moving charged particles produce a magnetic field perpendicular to the direction it’s spinning in. Protons in our body spin, which causes them to produce a small magnetic field as well.
The MRI machine is partly made up of a really strong magnet (something that produces a magnetic field). The magnetic field of this magnet forces the protons in your body to spin in the direction that produces a magnetic field parallel to the machine’s magnetic field.
While this is happening, a pulse of radiofrequency current (; a radio wave) is passed through the patient’s body. The radiofrequency forces the protons to spin in a different direction to align with its own magnetic field, away from the magnetic field of the magnet.
When the radiofrequency is turned off, the protons go back to aligning with the magnet. During this realignment, the protons release energy in the form of radio waves.
Interestingly, depending on the location of the water molecule in your body, the amount of time it takes for the realignment to complete, and thus the intensity of radio wave energy released, differs. An MRI sensor on the machine detects this energy to produce the gray-scale 3D model of the patient. The difference between radio wave intensity allows the differentiation of different types of tissues. Here’s a video visualizing how all this happens.
Unlike CT, MRI doesn’t use x-ray radiation, whose prolonged exposure could damage the cells.
Cell culture
To make tissues, we first need cells! In a lab, cells are cultured so that the first batch of cells proliferate to expand in population to create the next batches, and so on.
The cells to be used in the bioinks are grown in a culture medium that provides the essential nutrients for cell survival — amino acids, carbohydrates, minerals, vitamins. A commonly used culture medium is the Dulbecco’s Modified Eagle Medium (DMEM)… and I know what you’re thinking; relax, it’s not made from Eagle blood or anything😂.
Actually, DMEM contains salts that maintain an appropriate pH, the 13 essential amino acids, and vitamin B1, B2, B3, B4, B5, B7, B9, and Myo-inositol (another type of vitamin).
Other assistive materials such as growth factors and hormones are also mixed into the medium to modulate the behavior of cells. Particularly, the growth factors can be supplied in the form of a serum, most commonly the Fetal Bovine Serum (FBS), to signal the cells to grow. Unfortunately, the Fetal Bovine Serum isn’t just some funny last name as in DMEM. It’s a purified blood serum extracted from a fetus (unborn baby) of a slaughtered cow, so it has several ethical and cost issues. Also, since it’s extracted directly from living organisms, the concentration of different nutrients varies by the sample.
The cells, submerged in the culture medium, are allowed to grow inside an incubator, a closed, oven-looking environment that maintains an appropriate water concentration in the culture medium, temperature, pH, O2 and CO2 concentration, and humidity. The environmental conditions and nutrients provided depend on the type of cell being grown.
Bioinks
Biomaterial
Bioinks consist predominantly of biomaterials and cells.
Biomaterials (also called scaffold materials) are materials designed to directly interact with a biological system, like cells and the human body.
In tissue engineering, biomaterials are cell-friendly polymers that act as the artificial ECM that keeps the cells alive until they secrete their own ECM to form real tissues. More specifically, biomaterials serve four roles in tissue engineering:
- Keeping the cells in an aqueous environment. Biomaterials used in tissue engineering are usually hydrogels, which are a net of polymers that take in water to maintain a gel-like high-volume state. The water filling up the biomaterial exposes the cell to a water-based medium, which is similar to the internal environment of the body.
- Protecting the cells from damage, such as bursting, by the pressure during extrusion.
- Providing a surface for the cells to adhere to, which is essential for cell survival.
- Carrying nutrients and signaling molecules to keep the cells alive and help them undergo the tissue development process.
Mainly, 4 types of biomaterial — synthetic, natural, composite, and decellularized ECM — are used in tissue engineering.
- Natural polymers are derived from plants, animals, and algae. Natural biomaterials have good biocompatibility, along with lower risks of stimulating an immune response in the patient’s body. However, they generally degrade easily and are less resilient. Natural biomaterials used in bioinks include collagen (the structural protein in real ECMs), alginate (polysaccharide derived from algae), fibrin (tough protein partly making up blood clots), gelatin (a degraded form of collagen), and chitosan (derived from exoskeletons). Collagen and gelatin are the most commonly used because they contain RGD motifs (Arginine-Glycine-Aspartate amino acid sequence), which is a segment of the ECM’s adhesive protein that the cell’s integrin recognizes and binds to for cell adhesion.
- Synthetic biomaterials are artificially produced by chemical processes. They have good resilience and tunable stiffness but are less biocompatible and biodegradable (usually don’t degrade in cell-friendly conditions and produce harmful substances upon degradation). Commonly used synthetic biomaterials are PGA (poly(glycolic acid), material used in biodegradable sutures), PLA (polylactic acid), PLGA (polylactic co-glycolic acid, combination of PGA and PLA), PEG (polyethylene glycol, usually modified into hydrogel), and PEGDA (Poly(ethylene glycol) diacrylate).
- Composite materials are mixtures of natural and synthetic biomaterials, resulting in an intermediate material with enhanced biocompatibility and mechanical strength. A commonly used composite biomaterial is GelMA, composed of gelatin (natural polymer, good biocompatibility) and methacryloyl (MA).
- Decellularized ECM (dECM) is made by filtering out cells from an ECM of a functioning tissue and is the most biologically similar to ECM produced from cells. However, dECMs are harder to produce in large quantities and are more expensive.
When choosing which biomaterial to use, we have to keep three things in mind — biocompatibility, biodegradability, and mechanical property.
Biocompatibility
First of all, the biomaterial must have biological functionalities of the ECM, mainly supporting cell proliferation, adhesion, migration, and differentiation. To achieve biocompatibility, biomaterials often mimic the structure of natural ECMs.
The natural ECM consists of proteoglycans and adhesive proteins. Proteoglycans (proteo = protein, glycan = sugar) are a complex of structural proteins (ex. collagen) that provide the structural support and carbohydrate polymers called glycosaminoglycans (GAG). Adhesive proteins connect the ECM with the integrin protein in the cell membrane. The biomaterials serve the role of the proteoglycans — allow cellular messengers to pass through, provide a surface for cell adhesion, and store growth factors.
Proteins are sequences of molecular Lego blocks called amino acids. In a molecular level, cell adhesion occurs when the proteins called integrins embedded in the cell’s plasma membrane recognizes and binds to a particular sequence on the ECM’s adhesive protein. To allow cell adhesion, our biomaterial must have this sequence. Therefore, except for collagen, gelatin, and dECM, we often add a protein chain called the RGD motif to biomaterials to improve the biocompatibility of the scaffold.
The cells aren’t statically adhered to the ECM and remain in one position. Instead, the cells move throughout an organism’s life, most commonly when newer cells replenish the place of damaged cells. Cells move like skiers, where they temporary grab onto the snow with their poles and use that connection to pull themselves forward (+ downward). Instead of ski poles, cells produce a protein called focal adhesions to adhere to the ECM.
We call this movement cell migration, and to enable migration, the biomaterial must have interconnected pores that provide the hollow (water-filled) channels that the cells can travel through. The pores must have the shape and size that are large enough for the cells to move around in, but small enough to provide enough surface area for more cells to adhere to the biomaterial.
Besides being compatible with the cells in the engineered tissue, the biomaterial also shouldn’t provoke immune responses from the patient’s body.
Biodegradability
As the cells proliferate, they would expand through the biomaterial and take up the space once occupied by the biomaterial. Ultimately, all the biomaterial is meant to completely degrade and be replaced by the ECMs produced by the cells themselves.
Degradation happens in hydrolysis (hydro: water + lysis: to break). Essentially, a water molecule breaks a chemical bond by attracting individual molecules making up the bond and splitting itself to plug the ends of the newly broken bond.
Remember how biomaterials are hydrogels? Hydrogels are created when a liquid polymer precursor forms bonds between individual polymers and ultimately merge into one interconnected net.
When the biomaterial degrades, this process is reversed so that the polymers forming the net break off into their constituent monomers (smallest unit making up the polymer) and oligomers (smaller chains made up of a few monomers).
This degradation process must occur under biological conditions (temperature, acidity, pressure) favorable for cell growth, similar to the conditions inside the body.
When the biomaterial degrades, it is resorbed by the cells — they are broken down into their component molecules and reused inside the cells to make new cellular products. If the remnants of degradation (the monomers) are reactive, the cells would get damaged, so we need the biomaterials to degrade into cell-friendly material.
Although the biomaterial must eventually degrade, we don’t want it to degrade too quickly or slowly, so that it secretes native ECM just as much as it resorbs the biomaterial. At a restaurant, you wouldn’t pay 100 dollars to get a glass of water. The store owner also wouldn’t want to sell a 100% organic grass-fed steak for 1 dollar. It’s the same thing with the ECM.
Remember how biomaterials degrade when water molecules break off bonds between monomers by hydrolysis? Because hydrolysis is driven by water molecules, biomaterial degradation occurs in the presence of water.
Using the hydrolysis mechanism, we can control how quickly the biomaterial degrades in several ways:
- Hydrophilicity and reactivity. How strongly the molecules connecting the neighboring monomers attract and react with the water molecules in the medium.
- Surface area of the scaffold. The larger the surface area, the higher number of monomers are exposed to the cell medium for hydrolysis.
- Physical loading on the scaffold. Subjecting the scaffold under mechanical pressure like tension can increase the degradation rate by making the physical and molecular structure of the biomaterial less stable.
- Environment. Adjusting the scaffold environment, such as pH and temperature, and exposure to external energy sources like a magnetic field, electric field, ultrasound, and electromagnetic radiations (ex. ultraviolet, gamma) can speed up or slow down degradation. Caveat: the environmental condition should still support cell survival and growth.
- Catalysis by enzymes. We can further accelerate hydrolysis by using enzymes secreted by cells (a technique called enzymatic hydrolysis). For example, chitin, a natural biomaterial, can be degraded by an enzyme called lysozyme that assists that holds the neighboring monomers and distorts their bond to help with the hydrolysis.
Mechanical characteristics
We also want to be super-precise about the mechanical property of the biomaterial.
The biomaterial needs enough mechanical strength to withstand handling and surgical operations on the tissue. We can’t have a biomaterial that’s like burrata cheese and everything spills out the instant it’s incised (and yeah, I’m gonna keep using food analogies).
At the same time, the biomaterial must be soft enough so that the cells can freely migrate and proliferate. A tissue that’s too stiff would confine the cells to a certain amount of room they can expand to, and would also complicate blood vessels from the patient’s body trying to infiltrate into the new tissue to provide nutrients to the cells.
Most importantly though, the biomaterial must have the texture similar to the natural ECM in the particular tissue being made. As a tissue develops, stem cells use the mechanical property of the nearby ECM as information for what it’s going to differentiate into.
For example, they even measure the stiffness of the nearby ECM by applying force on it and measuring how much of it bounces back (I know, stem cells are so cute!!! 😍).
Although mechanical strength and stiffness might be used interchangeably in everyday language, they are distinct characteristics (just like how velocity and speed are two totally different things in physics!)
Mechanical strength is how much the material can withstand physical pressure and deformation and for how long. The mechanical strength is influenced by the type of bond — both crosslinks between polymers and within individual polymer molecules — holding scaffold together. Atoms are commonly bound together by covalent, ionic, or hydrogen bonds, in decreasing order of stiffness. Materials where all the constituent molecules are connected by covalent bonds have the highest mechanical strength.
Stiffness is how hard it is to deform the material; a rubber is less stiff than plastic because it takes much less strength for you to deform rubber than plastic. You can control the stiffness either by increasing crosslinking density, concentration, or molecular weight of biomaterial:
- To prepare the biomaterial for printing the scaffold, you mix the biomaterial with water to make a polymer precursor for hydrogels. Increasing the concentration of biomaterial increases the density of polymer molecules, and therefore stiffens the scaffold. It’s like making a dough; the more flour you add to the water, the harder the dough will become.
- You might know that an elephant moves less quickly than a mosquito. Why? Because it’s bigger and heavier. When a liquid flows, the molecules making up the liquid slid past each other, aka move. Since higher viscosity means the molecules are moving slower past each other, we can increase viscosity by increasing molecular weight, which makes it harder for the molecules to flow quickly. Increasing molecular weight is probably the most effective and safe solution, because you’re not severely restricting the area that the cells can move around in through crosslinking or molecular density. High molecular weight is another reason why natural polymers like alginate are commonly used for tissue engineering; they tend to have bigger molecules, meaning higher molecular weights
- Let’s talk about kidnapping. If you’re abducting someone, it’d be easier to kidnap them if you grab both of the victim’s hands than one hand, a leg in addition to the hands than just the hands, and all four limbs than all the previous options. In other words, the more parts of the victim you grab onto, the more you would restrict their movements. If you think of a polymer net in a biomaterial as kidnappers trying to kidnap each other by grabbing onto their neighbors’ limbs, the restriction of the individual molecules would increase with the density of the human-human linkages. Crosslinks are these chemical linkages in polymers that connect the kidnappers, and their density is proportional to the material stiffness.
Bonus section — parameters for deciding mechanical characteristic of biomaterials
Biomaterials are a type of polymer materials, or polymers. Many polymers experience a yield stress behavior, where experiencing a shear stress above a certain threshold — called a yield stress — causes the material’s behavior to change from solid to liquid. As I’ll mention later, the 3D bioprinters that extrude the bioink applies a force above this yield stress to let the material flow. Molecularly, the yield stress behavior is caused by the temporary physical crosslinked network between polymers that makes the material behave like a solid. The yield stress is the minimum magnitude of force required to break these physical crosslinks. This yield stress is higher for polymers with heavier molecules.
Crosslinking mechanisms
Let’s take look closer at crosslinks. During bioprinting, the liquid biomaterial hardens by a chemical process called crosslinking, where the polymer molecules making up the biomaterial form chemical linkages between each other into one integrated net.
When a biomaterial is still a hydrogel precursor that hasn’t been cured yet, the molecules making up the material are loosely linked to each other and slide past each other, which allows the liquid to flow. It’s like sand, which can flow, or be poured onto the ground because the individual sand particles aren’t bound together.
Crosslinking essentially integrates these sand particles into a rock, turning the liquid biomaterial solid. In the same way, crosslinking prevents the biomaterial from collapsing into a puddle of liquid and locks the individual molecules in place so they don’t get dissolved when the scaffold’s exposed to water.
The process that gets us from sand to a rock differs by which biomaterial we’re using. There are generally two types of crosslinking: physical and chemical.
Physical crosslinking is based on reversible physical interactions between molecules. There are three such interactions: ionic, stereo-complex, and thermal.
<ionic>
Ionic crosslinking occurs in water-soluble polymer molecules that are bound together by a common attraction to an ion of an opposing charge. Ionic crosslinking therefore requires a crosslinking molecule that, when dissolved in solution, would dissociate into an ion and an inert molecule.
Alginate is a polymer that crosslinks ionically. Alginate consists of alternating units of monomers mannuronic acid and glucuronic acid. The monomers have a high affinity for positively charged ions with charge +2 or +3, commonly a calcium ion (Ca2+). The addition of the crosslinker calcium carbonate (CaCO3) forms ionic bonds between calcium and the polymer molecules until the monomer chain coils into an insoluble gel.
<stereo-complex>
I have a confession to make. I have a finals coming up in a week, and two projects by the end of next week 😭, but I’ve been procrastinating and haven’t even started studying for half my tests. I’m getting pretty nervous, so it’d be great if you could pray with my right now that I’d get a decent grade for this semester.
So yeah, let’s pray. Dear god… 🙏
wait, no no no rewind.
Look what I just made you do. Interlock your hands together, right?
Imagine your hands were two molecules, forming a physical bond by complementary geometrical shapes. This is what stereo-complexes essentially are, a nets consisting of complementary molecules, such as those that are mirror-images of each other, clasped together.
Let’s take poly(lactic acid) (PLA), a synthetic biomaterial, for an example. There are two forms of PLA, let’s call them twin molecules, that have the exact same chemical composition but opposite atomic arrangements in reflection of each other. We call this reflective property chirality, and say that these two molecules are enantiomers (aka optical isomers) of each other.
To distinguish between the twins, we label one as L and the other as D. This gives us poly(L-lactic acid) (PLLA) and poly(D-lactic acid) (PDLA). When short strands each composed of PLLA and PDLA are mixed together, the twins lock together to form crosslinks and cure into a hydrogel.
<thermal>
Let’s say you’re hungry and want to fry an egg 🍳 (I’m actually getting hungry writing this article 🤪). On the hot frying pan, the egg you just cracked turns from clear to opaque, and by the time you have the egg on your plate it’s transformed from a slime to a solid.
The exact same thing happens with thermosensitive polymers, which change in solubility depending on whether the environmental temperature is above or below a certain point. Thermosensitive polymers can either have a Lower Critical Solution Temperature (LCST) or a Upper Critical Solution Temperature (UCST).
Thermosensitive polymers that become insoluble above a certain temperature have a Lower Critical Solution Temperature. Polymers that lose solubility below a certain point have a Upper Critical Solution Temperature.
For tissue engineering applications, thermosensitive polymer with LCST are mainly used, particularly those that are in liquid solution at room temperature, but become insoluble and separate from water above a certain temperature.
In the fried egg example, the unfried egg is really just proteins dissolved in water. When the temperature rises during cooking, the hydrogen bonds making up the protein’s 3D shape is broken and the proteins deform, exposing some hydrophobic parts of the protein and therefore making the whole protein insoluble in water.
Similarly, the increase in temperature breaks the transient associations holding up the polymer shape — hydrogen bond within and between polymers and the intermolecular bonding between hydrophobic groups — causing the polymer to collapse into a globule.
It’s almost like a balloon. If the balloon rubber were an agglomerate of polymer molecules and the air inside the balloon the hydrogen bonding, removing the air from the balloon would cause it to shrivel down. Since the polymer loses a support (hydrogen bond) holding up its shape, it condenses and becomes insoluble because the resultant globule has no room for the water molecules to penetrate through.
Chemical crosslinking is when polymers form covalent bonds between each other to form a permanent solid state structure. Three types of chemical crosslinking exist — wet chemical, enzymatic, and UV assisted.
<wet chemical>
Physical crosslinking was mainly based on electrostatic or morphological interaction between the polymer molecule, without any chemical additives. Chemical crosslinking use molecules called crosslinkers that form a bridge between neighboring polymers.
There are two types of chemical crosslinkers — homobifunctional and heterobifunctional.
Homobifunctional crosslinkers are molecules with two identical ends that allow the crosslinker to bind to two equivalent functional groups. They are mainly used for building up polymer chains by adding monomers one-by-one.
Heterobifunctional crosslinkers have two different ends, so they bind to two different functional groups. For this reason, they form crosslinks between polymer chains, and are more relevant to tissue engineering.
During “wet” chemical crosslinking, a hydrogel precursor is exposed to heterobifunctional crosslinkers to initiate the crosslinking reaction and quickly solidify the precursor into a gel.
<UV-assisted>
Some crosslinkers become active only when they are exposed to light, commonly UV radiation. UV radiation, like other types of light, are a type of electromagnetic radiation, a current of energy-carrying particles (called photons). When electromagnetic light is shown on a molecule, the energy from the radiation is transferred to the electrons of the molecule, making it reactive.
In the curing process, special crosslinkers called photoinitiators are added to the bioink. Upon exposure to light, photoinitiators decompose into reactive molecules that form crosslinks between polymer molecules.
Most often, ultraviolet (UV light) is used as the light source because it has the highest level of energy. However, certain photoinitiators called chromophores can absorb lights of lower energy (ex. visible light) and thereby allow the usage of other types of electromagnetic light.
<enzymatic>
Some crosslinking reactions are assisted by a biological enzyme. In fact, crosslinking is prevalent in cellular processes, most notably protein synthesis (ex. adding sugars, nucleotides, lipids, functional groups, forming bonds to maintain folds in the protein chain, combining individual proteins).
Several biomaterials mentioned earlier, like collagen, are proteins and therefore can take advantage of these enzymes produced from cells to accelerate crosslinking. Particularly, during catalysis, the enzyme helps connect the crosslinkers to the functional groups in the polymer molecules (see image below)
Each biomaterial has its own crosslinking mechanism, dependent on its chemical property, so you can’t randomly cure PLAs using ionic crosslinking (quiz: what crosslinking mechanism does PLA use?).
However, here are some advantages and disadvantages of physical vs chemical crosslinking (answer: stereocomplex crosslinking):
- Physical crosslinking is compatible with adding unstable molecules, like growth factors, to the bioink. Physical crosslinking is also safer for the cells, as cells aren’t exposed to potentially harmful chemicals or radiations.
- Chemical crosslinking occurs much quickly than physical crosslinking, and therefore decreases print time. Chemically crosslinking, since it’s permanent, produces a more stable solid structure with better mechanical strength.
Note: In tissue engineering, chemical crosslinking is usually used for stabilizing the printed structure, usually after physical crosslinking during printing, as part of the post-processing.
For both physical and chemical crosslinking, the crosslinking process can begin either as soon as the bioink is loaded onto the printer, once the ink is extruded, or after the whole scaffold has been printed.
The first method is when the crosslinker is mixed into the bioink, which may be preferred if the crosslinking mechanism involves an expensive crosslinker. Directly incorporating the crosslinker would minimize the amount of crosslinker used. However, since the crosslinking occurs in the entire batch of bioink even before the material is extruded, the overall viscosity of the ink would increase over time, which could make controlling the extrusion difficult.
For this same reason, the latter methods are overall preferred.
Type of cells
Here’s a question: what makes a cardiac muscle tissue? Yup, it’s because the tissue is composed of cardiac muscles cells 🧡.
The functionality of an engineered tissue really just depends on what cell lineages are embedded and in which patterns/locations. So, do we mix cardiac muscle cells into our bioinks to print cardiovascular tissue? Well, it’s not as simple as what you might think.
Scaffold-based tissue engineering relies on cell proliferation to amplify the initial amount of cells into larger numbers that eventually fill up the entire scaffold. However, fully differentiated cells (also called ultimately differentiated cells) and adult cells can only reproduce for a limited number of cycles, and we would end up with a sparsely populated cell scaffold that never turns into a working tissue.
Therefore, we instead use stem cells, which can reproduce much easily, and provide stimuli to the cells to help control what the stem cells differentiate into (more on this stimuli later). Multiple types of stem cells are used, with one stem cell giving rise to a more differentiated stem cell:
(Embryonic stem cells, although not used in tissue engineering, are at the top of the differentiation ladder and give rise to all the more differentiated stem cells explained below. They are only present in the embryonic stage of an organism and are the most flexible type of stem cell, producing all cell types besides the placenta and umbilical cord.)
- Adult stem cells (a.k.a. tissue-specific stem cells) are differentiated versions of embryonic stem cells that can produce any cell type in a particular tissue/organ. Adult stem cells are stored in small parts of certain tissues as backup cells that can replace damaged differentiated cells.
- Mesenchymal stem cells (MSCs) are cells with flexibility between pluripotent stem cells and adult stem cells. They are capable of producing a number of cell types, including bone, fat, cartilage, and immune cells. Mesenchymal stem cells are the most commonly used.
- Human-induced pluripotent stem cells (hiPSC) are ultimately differentiated cells that have been genetically engineered to reverse their differentiation and behave like embryonic stem cells.
- Progenitor cells, can differentiate into a few cell types within a tissue and stand in the middle ground between adult stem cells and ultimately differentiated cells. Unlike stem cells, they cannot reproduce indefinitely.
So… which one should we use? 🤯 It all depends on the level of invasiveness, cost, time, and the type of tissue you’re making:
- Adult stem cells, progenitor cells, and MSCs are collected invasively (by inserting surgical instruments into the person’s body) while iPSCs because they are grown from fully differentiated cells, are gathered noninvasively (ex. taking samples of your hair).
- Adult stem cells and progenitor cells are limited in availability in the patient’s (or the cell donor’s) body so only certain amounts of them can be collected at a time.
- Generally, the more differentiated the stem cell is, the easier it is to control the type of cell you get but also the fewer number of times that the cell can reproduce for.
Well, what if you want to use multiple types of cells at the same time? Love that question, a bonus point for you 🌟. Our tissues are complex entities, composed of multiple types of cells interacting with each other to enable the biological functions of the tissue. This means that your tissue isn’t always a Baskin Robbins gallon-sized cup, where discrete scoops of different ice cream flavors are contained in one cup (tissue);
rather, it’s closer to Dippin’ Dots, where a few cell types are mixed evenly, with the types of mixed cells varying by region.
Therefore, we occasionally need bioinks made of multiple types of cells, and we call them heterogeneous bioinks or multicomponent bioinks.
Cell origin
Besides the type of cell you are using, you must also consider the source of the cells. Collected cells can be either autologous (cells collected from the specific patient) or allogeneic (off-the-shelf cells derived from a healthy donor).
Allogeneic cells, also called off-the-shelf cells, can be used to make tissues for multiple other patients. Since only one source of cells is used, allogeneic cells are cheaper, less time-consuming to collect, and easy to produce at wider scales.
Autologous cells are specific to the patient, and therefore require extra time to collect and engineer the stem cells for each patient. Compared to allogeneic cells, autologous cells are less likely to stimulate immune rejection from the patient’s body, yielding higher success rates.
Cell concentration
The cell concentration in the bioink is determined by the cell type to mimic the environment of the native tissue as much as possible.
Cells use information about the cell density around them and the signals derived from neighboring cells to see if they should or should not proliferate. Too high cell concentration would signal to the cells that they should stop proliferating (a phenomenon called contact inhibition). Too low cell concentration, on the other hand, would also inhibit sufficient cell growth due to the lack of enough growth factors produced by neighboring cells.
Cell concentration also affects the overall metabolic rate of the scaffold. High cell concentration requires a higher supply of oxygen and nutrients at higher frequencies. Furthermore, since nutrients come from the physical environment outside the scaffold, high cell density renders it difficult to get nutrients to cells deeper inside the scaffold. The byproducts of cell metabolism, called cell waste, accumulate much more quickly with higher amounts of cells and require a removal system.
In addition, bioink viscosity is proportional to the cell concentration. Viscosity is how much mechanical force is needed to get the bioink to flow. Low cell concentration corresponds to low bioink viscosity, which is easier to extrude because less mechanical force is needed to extrude them out. High cell density bioinks can cause nozzle clogging and requires higher mechanical force for extrusion, which may damage the cells and affect cellular behavior.
Printing method
Once all the bioink have been prepared, they are deposited into the desired 3D structure by 3D bioprinting. Three main types of bioprinting are currently used: extrusion-based, inkjet-based, and light-assisted.
Extrusion-based bioprinting
Extrusion-based bioprinting uses an extruder to mechanically push bioink out from the cartridge through the nozzle. In this way, the bioink is extruded onto a flat surface in continuous filaments, building the 3D structure layer-by-layer.
Extrusion-based printing is the most common method used for fabricating bioscaffolds due to their feasibility, flexibility, and relatively low cost.
One problem with extrusion-based bioprinting is that you must wait for each layer to crosslink before extruding the next layer so that the printed structure won’t collapse. Because of this issue, a bioprinting technology called FRESH (Freeform Reversible Embedding of Suspended Hydrogels) has been getting traction. Essentially, the bioink is extruded into a tank full of granular gel (the texture of hair gel) made from hydrogel. The granular gel, called the support bath, holds the bioink in place until it cures and essentially acts like a mobile mold. You can read more about FRESH here.
Inkjet bioprinting
Inkjet bioprinting is fairly similar to extrusion-based bioprinting, except for the extrusion process. Essentially, the bioink stored in the cartridge, normally held in from falling through the nozzle by surface tension, is released in droplets by pressure pulses caused by thermal, electrostatic, or piezoelectric actuators that allow the droplet to overcome surface tension.
Thermal actuation
A voltage pulse locally heats up the bioink, forming a bubble that subsequently pops to generate a pressure that pushes a droplet of bioink out from the nozzle.
Piezoelectric actuation
Upon a voltage pulse, the ring-shaped piezoelectric actuator surrounding the cartridge is squeezed, which changes the volume of the cartridge and creates a wave of pressure transferred down to the nozzle to allow the droplet to overcome the surface tension.
Electrostatic actuation
A voltage pulse causes the actuator to bend as to expand the volume of the cartridge, which induces a pressure wave as in the piezoelectric actuation.
Since no artificial mechanical force is pushing out the bioink as in extrusion-based bioprinting, the cells aren’t subjected to harsh pressure that could damage them. However, inject printing is limited in certain bioink viscosities it can print and thus is less flexible than the extruder-based bioprinting technique.
Light-assisted bioprinting
Light-assisted bioprinting generally uses radiation of light to crosslink the bioink. The most common type of light-assisted bioprinting technique is Stereolithography (SLA), where a beam of laser scans over a tank filled with photocrosslinkable liquid bioink, selectively curing the bioink layer by layer. The resulting structure is then processed to remove the uncured, excess bioink remaining on its surface.
The bioink used in light-assisted bioprinting utilize UV-assisted crosslinking mechanisms, where special crosslinkers called photoinitators are incorporated into the bioink and form crosslinks when enacted by illumination.
Light-assisted bioprinting allows for high-resolution printing because the liquid tank of bioink holds the 3D structure in place during crosslinking so that it won’t collapse as in printing in the air (similar to the support bath used in FRESH bioprinting). However, this technique is slower, less material-efficient (all the leftover liquid bioink is wasted!), limited to bioinks containing biomaterials that cure by exposure to light (called photopolymers), and may, in turn, result in cell damage when the bioink is exposed to intense high-energy radiation ( → cancer 🦀). Light-assisted printing is also less compatible when printing with multiple bioinks.
Generating the 3D printing image
All 3D printers above build their 3D structures based off of a 3D image that gives the printer instructions on in what direction to move, how quickly to move, when to extrude the print material, and when to stop.
Scaffold Culturing
So, now our tissue scaffold is printed, we just have to implant it in the patient’s body and it would be all good, right? Again, I hope it was that easy, son 😕; go blame your mommy nature 🌳.
After the engineered tissue scaffold is bioprinted and fully crosslinked, it’s still a baby and doesn’t know what it’s actually going to become 👶 or what to do. You have to feed it with some more nutrients and hormones (ex. parental pressures ☹) so that it can perform biological functions and survive in the patient’s body without assistance in a process called tissue maturation/functionalization. We can help the tissue mature in two ways — biochemically and mechanically.
Biochemical stimuli
Previously, I mentioned that stem cells are used for tissue engineering. These stem cells can either be differentiated before they are mixed into the bioink, or directly incorporated into the bioink as stem cells.
In the latter option, we can induce the stem cells in the scaffold to undergo differentiation into specific cells by adding special supplements to the culture medium. For example, Mesenchymal Stem Cells (MSC) can be induced to differentiate into bone cells (osteocytes) by culturing the MSC-containing scaffold in an osteogenic medium.
Other than cell differentiation, cells gather information to guide their cellular behavior from their environment, namely the ECM that surrounds them. The ECM harbors cell-directing molecules and peptides that signal the cells to carry out specific tissue development processes, such as morphogenesis (spatial arrangement of different types of cells) led by chemotaxis (movement of cells in response to signals from the ECM).
Likewise, such signaling molecules, serving as the biochemical stimuli, can be incorporated into the scaffold material by suspension (physically mixing them with the biomaterial) or bonding with polymer molecules making up the material (chemically combining them with the polymer). As the biomaterial degrades, the biochemical stimuli would be released and taken up by the cells to regulate their behavior. This also means that, we can control the degradation rate by adjusting the bioink viscosity and in turn the scaffold stiffness.
Alternatively, biochemical stimuli can also be mixed into the cell medium, so that they are present from the beginning of the cell functionalization process but not when the scaffold is implanted onto the patient. This option would be sufficient for most nutrients/signaling molecules, but you may want to keep the cells under continual exposure to certain molecules by incorporation into the scaffold.
The most commonly used signaling molecules include Transforming Growth Factor beta (TGF-beta), which are multifunctional growth factors that guide cell differentiation, proliferation, and chemotaxis (fun fact: TGF-betas are naturally secreted by white blood cells).
Additional factors like growth factors, angiogenic factors (drugs encouraging angiogenesis into the engineered tissue, including vascular endothelial growth factor (VEGF)), and immunosuppressive factors (elements that inactivate the immune system to prevent tissue rejection), can be added to the medium to help regulate the interaction between the engineered tissue and the patient’s body.
Biophysical stimuli
The cells are also affected by the mechanical and morphological properties of their surroundings, called biophysical stimuli.
Biophysical stimuli divide into static and dynamic stimuli.
The static stimuli — including biomaterial stiffness, elasticity, and surface morphology — are unchanging and embedded in the bioscaffold. These properties vary by the biomaterial being used, crosslink density, bioprinting resolution (how fine the details of the 3D construct are), and the 3D model being printed.
Dynamic mechanical stimuli on the cells are applied during tissue functionalization and include the pressure of fluid flowing through the scaffold (called hydrostatic pressure), shear stress, atmospheric pressure, and exposure to external sources of energy such as the electric field, magnetic field, ultrasound, and light.
Unlike biochemical stimuli, whose molecular interactions with the cells are relatively well-explored, the effect of biophysical stimuli on cell behavior is less known.
Usually, scaffolds are matured in a cell medium inside an incubator (the magic cell oven that you saw in the cell culture step). The incubator controls conditions like O2 and CO2 level, temperature, pH, pressure, and humidity, and the scaffold just sits there until the cells proliferate, differentiate, adhere to each other, and replace the scaffold material with their own ECM. Thereby, the cells are only exposed to biochemical stimuli. Growing the tissue scaffold in this way is called static cultivation.
There are several problems with static cultivation. Mainly, static cultivation depends on nutrients and oxygen to reach the cells in the scaffold by slow diffusion, which causes only cells in certain regions of the scaffold to get the nutrients. Also, the cell medium has to be manually exchanged to replenish the nutrients and remove cellular metabolic waste.
Recently, an alternative way for functionalizing the tissue — you guessed it — dynamic cultivation has been gaining traction, where a super cool cell oven (let’s say, with the additional functionality of slapping you when you say a curse word), called a bioreactor, controls the environmental conditions as well as the flow rate of the cell medium and biophysical stimuli.
So how does the bioreactor work? Bioreactors are all intended to mechanically mimic the internal environment of the body and supply the culture medium to all cells in the scaffold. Therefore, the type of bioreactor used depends on the type of tissue, and several types of bioreactors have been developed:
Spinner flask bioreactor
In short, spinner flask bioreactors are hand mixers in a glass bottle.
The spinner flask consists of two arms, tissue scaffolds threaded through needles sticking out from the, and either a stir rod (the first figure below) or a shaft stirrer (second figure) that consistently spin to mix the cell medium and thereby keep an even supply of oxygen and nutrient throughout the medium.
I’ve seen a shaft before, but what the heck is a stir rod?
If you’ve played with magnets before, all magnets have two poles. Two opposite poles tend to attract each other — let’s call them Romeo and Juliet — and this attraction creates a magnetic field that determines how a nearby magnet (or a moving electrically charged object, not relevant to the bioreactor) would move. A magnetic field is analogous to the sense of love/hatred, where the closer you are to your crush, the more you want to walk towards them. When you encounter your ex-lover who cheated on you, you would move further away from them.
If two magnets are separated by a layer of glass, rotating one magnet would cause the other to rotate as well because the magnetic field causes the Romeo of one magnet to pull the Juliet of the other magnet along with it and vice versa. Essentially, rotating a magnet causes its magnetic field to rotate as well, and in turn, cause the object the magnetic field is influencing to rotate.
So what? Congrats! You just learned how a stir rod works. Basically, a stir rod is a piece of magnet coated in a chemically unreactive material that spins quickly in response to the change in the magnetic field caused by another rotating magnet placed under the bioreactor flask. The spin of the stir rod causes the cell medium to mix and evenly distribute nutrients to the cells.
A stir rod stirring a liquid in a beaker. Outside of bioreactors, stir rods are used for mixing solutions in laboratories.
Although spinner bioreactors have been widely used, they produce regions of the scaffold under high shear stress, especially near where the stir rod or shaft spins, which may damage the cells.
Rotating-wall vessel bioreactor (rotary bioreactor)
The rotating-wall vessel bioreactor consists of a cylindrical vessel containing cell media and tissue scaffolds, a motor for rotating the vessel, and a membrane for the exchange of CO2 for O2.
The bioreactor continually rotates horizontally (along with its height), and the rotation creates a shear force by the rotational flow of shear medium and centrifugal force (the outward force enacted on an object spun in a circle around an axis). These two forces cancel each other out so that the cells experience only gravitational force, and no other force (if you were the scaffold you would feel like you were falling in the air the whole time, called a free-falling state).
The free-falling state encourages the scaffold to grow uniformly, without inflicting high shear force in local regions as in the spinner flask bioreactor. For the same reason, this bioreactor is adequate for growing fragile tissues, as the scaffolds are subjected to minimal mechanical force, including shear stress.
Perfusion bioreactor
The two previously bioreactors, spinner flask, and rotating-wall vessel bioreactor, allow for even mixing of nutrients into all parts of the cell medium but aren’t specialized for allowing that medium to perfuse to all the cells in the scaffold. Well, here’s where perfusion bioreactors shine ✨
Perfusion bioreactors constantly pump the culture medium into the tissue scaffold in a cycle. They consist of the media reservoir to store the cell medium, perfusion chamber (container for the scaffold where the culture medium passes through the scaffold), and tubing and a pump to force the medium from the reservoir through the scaffold and return the medium that has passed through the scaffold back to the reservoir.
The perfusion bioreactor can either pump the liquid medium directly or indirectly, depending on the scaffold size.
In indirect perfusion bioreactors (figure A in the image below), the scaffold is loosely held in the perfusion chamber so that some room is left between the scaffold and the walls of the chamber. This extra room causes much of the cell medium to pass through the space between the scaffold and the chamber walls. Thereby, the perfusion rate of the medium mainly controls the nutrient concentrations near the surface of the scaffold. This makes indirect perfusion bioreactors more fit for functionalizing smaller tissues, which have a relatively larger surface area to volume ratio.
Direct perfusion bioreactors (figure B), on the other hand, target larger tissue scaffolds, as the scaffold is fit exactly through the cross-section of the perfusion chamber and all the cell medium must pass directly through the scaffold.
Compression bioreactor
Compressive bioreactors are exactly as they sound; they apply pressure to the tissue scaffold.
Compressive bioreactors consist of one or more pistons, a motor to move the piston up and down, and a compression chamber containing the scaffold and cell medium.
The piston is either pushed into the compression chamber and remains there to create a prolonged state under pressure or consistently pushed in and out to mimic the cycles of pressure loading and unloading on tissues like bone and cartilage.
Hey Yelim, so when exactly is the tissue ready to be implanted in the patient? Is it when all the biomaterial is replaced by the native ECM?
Actually, there’s no clear answer to that question yet (and yes, this is also what makes the field of tissue engineering so exciting!). For now, we can only say that the tissue would be implanted as soon as the tissue is mature enough to survive in the patient’s body, meaning that the tissue must have the mechanical stability to not disassemble or tear in the body and perform biological functions to cure (and not just treat) the disease caused by tissue damage.
Challenges in tissue engineering
Tissue oxygenation
We all need to breathe oxygen to survive. But why does our body need it? Oxygen is an essential substance that serves a variety of functions in the cell. For example, it is an essential component of cellular respiration, a chemical chain reaction that produces energy from sugar. Also, oxygen guides tissue formation processes, like forming of new blood vessels and ECM synthesis.
Lack of sufficient oxygen supply leads to a condition called hypoxia, which, when prolonged, leads to cell death. Therefore, tissue oxygenation is vital for the survival and functionalization of the tissue.
Some dynamic tissue functionalization methods, as mentioned earlier, can help with getting oxygen to reach cells deeper inside the scaffold. However, the real problem comes after tissue implantation. After the engineered tissue is surgically attached to the patient’s body, it no longer has access to the artificial sources of oxygen as it did inside the incubator or bioreactor. To obtain oxygen and manage its cellular waste, the tissue must wait for the patient’s blood vessels to penetrate into it (quiz: what’s this process called? …
Oxygen diffuses from the blood vessel called capillary into the neighboring cells for tissue oxygenation
answer: angiogenesis). However, it takes about 14 DAYS for angiogenesis to be completed, so we need a way to keep the tissue alive and minimize hypoxia during these two weeks after implantation. Two major methods are being developed to tackle this challenge.
First, you can embed artificial blood vessels throughout the scaffold by bioprinting thin channels, called microchannels, into the tissue scaffold and later seed the microchannels with endothelial cells (cells covering the inner surface of blood vessels). Having artificial blood vessels accelerate angiogenesis and also helps with even oxygen diffusion during tissue functionalization, even when using static functionalization methods.
You could also take advantage of symbiosis with photosynthetic microorganisms (photosymbiosis), particularly microalgae. Bioinks containing algae cells can be bioprinted with human cell bioinks to serve as a natural oxygen provider during tissue functionalization, and even after implantation (the latter idea hasn’t been tested yet). Using algae for mammalian cell oxygenation has been explored in other medical fields, like tissue regeneration (helping regrow damaged tissues).
For example, a study by Chavez et al. embedded a bioscaffold made from algae bioinks onto a scar on mice for 14 days to help with wound healing. By oxygenating the cells on the site of the wound, the algae helped with angiogenesis and collection of endothelial cells onto the wound, both of which indicate progression of wound healing. Interestingly, the algae didn’t stimulate any immune responses from the mouse’s immune system, and other studies on photosymbiosis between algae and mammalian cells also revealed no or minimal immune responses against the photosynthetic organisms. (Article on photosymbiosis coming soon!!)
Tissue functionalization
Before we even get to worry about tissue oxygenation after implantation, we first have to address our biggest problem: getting the tissue to actually work.
Right now, our tissue engineering researchers are jealous bakers trying to figure out a recipe to the world-renowned chocolate cake from a neighboring bakery.
They know what cake they’re supposed to get at the end and have the all the tools they need — whisks, stainless bowls, spatulas, oven, piping bags, but don’t quite know what ingredients at which ratios and procedure they should mix them into the perfect cake.
We do have the bioprinting and bioreactor technology to fabricate functional tissue. However, we are still uncertain as to how the cells behave and interact with each other and their ECM to create a biologically functioning tissues.
To find out this secret recipe to tissues (yum!), researchers are using monitoring techniques, in combination with computer simulation programs and artificial intelligence, to better understand this mechanism.
Once we figure that out, we would be able to adjust the types of cells, biomaterials, cell medium, growth factors, and mechanical stimuli to make real tissues that save lives.
TL;DR
- Globally, patients are suffering due to the shortage of organ donations and the effects of rejection of the donated organ by their bodies. This issue demands a way to custom-engineer tissues and organs from the bottom to top.
- Tissue engineering is a field working on engineering functional organs that can replace the function of tissues that have been damaged beyond repair.
- In a body, tissue development happens over a span of multiple complex cellular processes, such as cell proliferation, differentiation, migration, extracellular matrix (ECM) formation, and angiogenesis. The extracellular matrix heavily determines the behavior of the cells by providing signaling molecules and nutrients.
- Tissue engineering involves fabricating brand-new tissues from cells embedded in tissue scaffolds, porous structures that mimic the function of the ECM.
- Tissue engineering consists of 6 major steps: (1) obtaining the 3D model of the scaffold to be fabricated, (2) culturing the number of cells to be used to make the scaffold, (3) creating bioink solutions (mixtures of cells and biological polymers that are extruded into tissue scaffolds), (4) loading and printing the bioink into scaffolds using bioprinters (type of 3D printer), (5) culturing the scaffold into a biologically functioning tissue, (6) implantation of the engineered tissue into the patient’s body through surgery
- Current challenges with tissue engineering include supplying enough oxygen to all cells in the scaffold during scaffold culturing and after implantation and getting the tissue to autonomously work like the normal tissues in our body
Works Cited
- https://www.closerlookatstemcells.org/learn-about-stem-cells/types-of-stem-cells/#mesenchymal
- https://www.allevi3d.com/choosing-the-right-cell-number/
- https://www.sciencedirect.com/science/article/pii/S0142961216302666?via%3Dihub
- https://www.sciencedirect.com/science/article/pii/S2590006419300146
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069718/pdf/ijms-22-03971.pdf
- https://www.sciencedirect.com/science/article/pii/B9780128239902000064
- https://www.researchgate.net/publication/327558886_Review_on_bioreactors_in_tissue_engineering/references#fullTextFileContent
- https://www.researchgate.net/publication/272660002_Bioreactor_Systems_for_Human_Bone_Tissue_Engineering
- https://pubmed.ncbi.nlm.nih.gov/24547761/
- https://www.khanacademy.org/science/ap-biology/chemistry-of-life/introduction-to-biological-macromolecules/v/hydrolysi s
- https://www.sciencedirect.com/science/article/pii/S0142961216302666?via%3Dihub
- https://www.nature.com/articles/nmat2441
- https://www.cancer.gov/about-cancer/diagnosis-staging/ct-scans-fact-sheet
- https://www.youtube.com/watch?v=l9swbAtRRbg
- https://www.sciencedirect.com/science/article/pii/S0169409X03000425
- https://www.sciencedirect.com/science/article/pii/B9780857097132000018
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1121941/pdf/35.pdf
- https://www.mechanobio.info/development/what-is-cell-migration/
- https://theory.labster.com/ionic-crosslinking/
- https://www.intechopen.com/chapters/68305
- https://info.gbiosciences.com/blog/the-3-types-of-crosslinking-reagents-and-when-to-use-them
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546294/
Personal remarks
First, I’d like to give a shoutout to Dr. Jaci Bliley, Dr. Adam Feinberg, and Dr. Dan Shiwarski (super cool people who also developed the Freeform Reversible Embedding of Suspended Hydrogels bioprinting technique) who helped me get a deeper understanding of tissue engineering.
Although we do have some more progress to be made, getting to the point of assembling cells into fully-functioning organs in the future would change the world — both in hospitals and laboratories.
Imagine a world where no one had to die waiting for an organ transplant, and no, you also wouldn’t have to sacrifice animals just for carving out one organ from their body. This would also be a world without organ trafficking, no organ rejection, and no “temporary treatments”.
Tissue engineering could also be used as biological models to better study cellular behavior and test new drugs more accurately and without 111 MILLION mice being sacrificed every year as lab specimens. Although we wouldn’t be able to completely eliminate the dependence on lab rats right away, organ engineering would certainly expedite humanity’s progress towards longer and healthier life.
My name is Yelim, an innovator and builder passionate 3D/4D Bioprinting and soft robotics. Check out my socials below or connect with me through email: yelim.kim0229@gmail.com.
I’ll be writing about how algae can be used to improve tissue engineering and tackle its major problems, so make sure to come back for that:) I look forward to seeing you again, and please leave a clap or comment on this article (bonus point if you do both)!
