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Gabrielle Maranga Chemical Information & Retrieval Dr. Bradley Fall 2011

Three-Dimensional Printing: The Future of Medicine

The cosmos, the Mariana trench, Grand Canyon; what could three things so vastly different have in common? Each of these represents the epitome in each of their respective dimensions—height, width and depth. Though height and width are challenged almost every day by architects and engineers, finding ways of redefining depth (outside of nature) can prove difficult. However, it’s this vital third dimension that gives brings our otherwise flat world to life. Bringing depth into something as simple as a cartoon allows animators move beyond a drawing on a paper and bring their characters to life in model space using highly realistic computer generated images. In a similar vein, although the process of printing has been modernized greatly since Guttenberg’s press, the output has always been the same: a sheet of paper containing words or images. However, after over 500 years of modern advances, the concept of printing has brought into the future. Three-dimensional printing (3DP) incorporates depth, this invaluable third dimension, into traditional printing, allowing solid, customizable objects to materialize out of thin air. The only limit to this technology is the boundary of our imaginations.

The ability to create extremely detailed and precise objects has boundless applications in engineering, aeronautics, cosmetics, nanotechnology, biotechnology and architecture. An article appearing in the February 2011 edition of the Economist titled “Print Me a Stradivarius” summed up the impact that 3DP will have on the world by saying “Just as nobody could have predicted the impact of the steam engine in 1750—or the printing press in 1450, or the transistor in 1950—it is impossible to foresee the long-term impact of 3D printing. But the technology is coming, and it is likely to disrupt every field it touches [1].” As traditionally noted, the editor and corresponding reporters for The Economist are kept anonymous.

Even more recently, Stratasys, a 3D printing company has a full page ad featured in The Wall Street Journal with the tag line, “Whatever your game, 3D Printing is going to change it [2],” as seen in Figure 1. The ‘game’ that has the most possibility to be changed is the medicinal discipline. By its very nature, the medical field is constantly absorbing and incorporating new ideas and concepts to further advance the capabilities of the human body. Building up from the most minuscule level to visible physical reality, 3D printing will have an effect on every aspect in the medical field. On the molecular level, 3D printing has the potential to revolutionize the pharmaceutical world in creating new drug delivery systems; by using stem cells instead of substrate, 3D printing is able to produce functioning organs eventually ready for transplanting; and finally on the large scale it has the ability to print fully operational prosthetics.


 * Figure 1: Wall Street Journal Ad featuring Three Dimensional Printing [2]**

Initially, 3D printing was developed as a way to produce fast and cost-efficient trial samples of complicated models [3]. The umbrella title for this field is called rapid prototyping (RP). While there are different variations, the three main types of rapid prototyping machines are stereolithography, (SLA) [4], selective laser sintering (SLS) [5], and three-dimensional printing (3DP) [6]. Each method utilizes different ways of producing a solid object. A schematic of the different types of rapid prototyping is exhibited in Figure 2 [7].
 * Figure 2: Different types of Rapid Prototyping [7]**

SLA principally uses ultraviolet radiation to polymerize a liquid solution of almost any material into the solid shape desired. The UV light is applied orthogonally to a bed of the material to be solidified. This method can only create one x-y plane of the object at a time. This plane is then lowered to a predetermined amount on the z-axis and the next viable layer of product has the UV light applied to it to form the next additive segment. A good analogy for this method is to think of a pottery wheel. Each layer of clay depends on the previous layer beneath it and the piece builds structurally from the bottom towards the top. Also following the analogy, no extra material is used to change the wet clay into its hardened form; it is baked in a kiln which is analogous to the UV light being applied to the material.

Selective laser sintering uses a powerful laser to make a cohesive piece out of powder through a process called sintering. The method of sintering happens when the powder is heated to a temperature beneath its melting point, but that is high enough so that the particles can diffuse across the boundaries and become one continuous solid [8]. The heat source in this set up is that of the laser applied to a bed of powder. The laser follows the output of the CAD description of the object and prints each layer onto a fresh layer of powder. Continuing with the art comparison, SLS would be more akin to the “magic sand” that was a popular childhood toy [9]. Instead of using heat from a laser source, the magic sand becomes a solid structure when it comes in contact with water, almost like ‘reverse-sintering.’ With SLA and SLS, the types of material that can be used are innumerable. This benefit, called local composition control (LCC), allows the manufacturer to use almost any product available; rubber, metal, silicon, and plaster are just a few examples. With LLC, a separate binding solution to form a cohesive solid is not required, as is exhibited in 3D printing.

The third type of RP, and arguably the most versatile for the medical field, is three dimensional printing. Three dimensional printing was patented at the Massachusetts Institute of Technology in 1992 by Emanuel Sachs, John Haggerty, Michael Cima and Paul Williams [6]. 3D printing quickly traversed into the pharmaceutical realm. Instead of using lasers or ultraviolet lights, this process emulates an ink jet printer. A very thin layer of drug blend (active drug and excipients) is rolled into the processing bed, analogous to the paper in a printer being fed under the print head. Here, the print head moves in accordance with a previously programmed image, usually a computer aided design (CAD) drawing. Instead of ink, the print head extrudes a binding solution, which is unique to each drug created as it must chemically interact with the excipients present to form a polymerized solid dosage form when properly dried. When an ink jet printer finishes printing a ‘page’, it retrieves a new blank page and continues the printing process. The 3D printer prints horizontally rather than vertically. The entire processing bed is lowered into the z-dimension and a new layer of drug blend is rolled onto the previous layer. The process is then repeated until the object is built from the bottom up. RP techniques are similar in that each layer of the product is created one x-y plane at a time until the preset depth z is met. A visual demonstration of this process is shown in Figure 3 [10]. Much like integrating a curve in calculus by adding up rectangles of infinitely small widths; the final object is formed in 3D printing by adding planes of infinitely small depths on top of each other.
 * Figure 3: Visualization of the three dimensional printing process [10]**

D. Dimitrov et. al, categorizes the types of binding solutions that can be used into two subsets, “continuous ink jet printing (continuous deposition) [which] uses a stream of charged droplets and deflects those, which are to be used for printing [and] drop-on-demand (DoD) ink jet printing [which] positions the ink jet printing head over the place where printing is to occur before depositing a droplet. [11]” In some cases, the specific material that is being used to build the prototype is dropped from the printer, a “liquid-to-solid” compound, so that each drop is being dropped on a site where a previous drop was located in a prior layer. Binders like this have additive properties and this technique is thusly called “drop-on-drop” 3D printing. The type of DoD printing used in pharmaceuticals is called “drop-on-bed” printing, where the binding fluid is dropped onto a bed of fresh (not bound) drug blend. This method allows for the binder to interact with a new layer of excipient every time instead of interacting with the previous coating of liquid binder, which allows for a stronger bond between the products.

The tablets that are produced from three dimensional printing are a novel new drug delivery system. The different properties can be developed and uniquely customized to enhance many different applications. Since they are made primarily of loosely bound active drug and fast dissolving excipients, these tablets dissolve almost instantly when placed in the patient’s mouth, eliminating the need for water and large pills. As Mizumoto et al. summarized, “The most desirable formulation for use by the elderly is one that is easy to swallow and easy to handle. Taking these requirements into consideration, attempts have been made to develop a fast-disintegrating tablet...Since such a tablet can disintegrate in only a small amount of water in the oral cavity, it is easy to take for any age patient, regard- less of time or place. For example, it can be taken anywhere at any time by anyone who do not have easy access to water. It is also easy to dose the aged, bed-ridden patients or infants who have problems swallowing tablets and capsules [12].”

The limitless architecture of tablets printed using 3D printing expands the market of pharmaceutical drugs available. Taking full advantage of the porous properties of the excipients utilized in these fast dissolving tablets, Rowe et al. developed four types of gradient or controlled release situations that could be created by 3D printing. The first, termed “immediate-extended.” Much like a conventional pill this tablet is absorbed in the stomach as soon as the drug is available. The second is a “breakaway” tablet. These pills contain two drug loaded centers coated with a soluble phase which eventually disintegrates to release the sub-units. The third is an “enteric dual pulsatory” tablet which is treated with a polymer coating that only allows it to dissolve in the intestines. Similar to the breakaway tablet, this drug delivery system also contains two drug loads so that the prescription is released twice. Finally, the fourth type of tablet is a “dual pulsatory,” which has two sections, each with different pH solubility. This chemical difference allows the drug to be absorbed in two locations, first in the gastric fluid and subsequently in the intestinal fluid [13].

One of the best analytical tests to determine the efficiency of tablets with these qualities is a dissolution procedure. the 3DP method is testable using a dissolution procedure. Through mechanical agitation in a liquid environment created to simulate specific in vivo conditions (pH, fluid composition, etc.), the assay of the removed aliquots are taken over time to determine what quantity of the drug load is released. Wu Weigang et. al produced a paper that showed the dissolution results of testing in vivo with controlled release drugs produced by three dimensional printing [14]. The two drugs studied were levofloxacin and rifampicine. These drugs are typically used to treat diseases associated with chronic osteomyelitis and bone tuberculosis. Graph 1 shows the dissolution results found for each drug within the bones, blood, and muscle of rabbits. As Weigang’s team hoped, only the levofloxacin appeared in the bones and muscle of the rabbits over the first seven days. The absence of the drug in the blood meant that the levofloxacin was not being excreted from the body and that it was actually being used. The presence of rifampicine was not detected until the eighth day, as both drugs remained in relatively high and constant concentrations for approximately six weeks. The remarkable outcomes of this trial alone show only some of the possibilities of this process. The need for constant and long-term drug therapy treatment can be eradicated for some patients through 3D printing technology because this type of drug implant can be easily created and manufactured.


 * Graph 1: Levofloxacin and Rifampicine In-Vivo Testing [14]**
 * Graph reproduced with data presented Weigang's article**
 * Concentration units for bone and muscle are ug/g, units for blood are ug/mL**

Three dimensional printing is an ideal candidate for the future of pharmaceuticals, having more versatility than other rapid prototyping methods such as SLA and SLS. Since other RP methods necessitate lasers to provide ample heat sources for binding products together, they can not be used in the pharmaceutical field. Many of the chemical molecules used in pharmaceuticals are heat sensitive, which makes 3D printing superior in this respect.

Beyond pharmaceuticals however, 3D printing is taking leaps and bounds in the realm of tissue engineering. Until very recently, the thought of using 3D printing in bioengineering has only been an idealistic dream. Very recently, however, there have been great advances in this niche field. Earlier this year, a paper was published by T. Xu, et. al that simultaneously printed a sample containing three distinct cell types: human amniotic fluid-derived stem cells (hAFSCs), detrusor smooth muscle cells (dSMCs), and bovine aortic endothelial cells (bECs) [15]. While scaffolds have been printed using natural polymers and plaster materials [16], this research utilizes biologically acceptable materials.
 * Section 2: Tissue Engineering**

However, earlier last month, a team of research scientists lead by Faulkner-Jones, et al. from Scotland's Heriot-Watt University published a journal article in //Biofabrication// showing that it is possible to print viable tissue using human embryonic stem cells (hESC) [17]. Using a dual printing method of cross-printing the stem cells suspended in medium while simultaneously printing solely the medium allowed these researchers to create a gradient of hESCs in the well plates, as seen below in Figure 4. When these well plates are inverted, the collection of hESCs aggregate and begin to form clusters of cells. Detailed microscopic views of these aggregates can be seen in Figure 5, both 24 and 48 hours after a successful printing (the images are grouped in three columns).


 * Figure 4: Aggregates of hESCs printed via 3D Printing [17]**


 * Figure 5: Microscopic Views of Cell Aggregates After Printing [17]**

As 3D printing has a high level of accuracy in positioning these cells, as in pharmaceuticals, the architecture of these cell groups and tissues are limitless. As Faulkner-Jones concluded, "This work demonstrates that the valve-based printing process is gentle enough to maintain hESC viability, accurate enough to produce spheroids of uniform size, and that printed cells maintain their pluripotency [17]." Due to the highly specific and non-invasive nature of 3D printing, the hESCs maintained a high percentage of viability and most importantly, their ability to emulate and create any type of human tissue. Based on the images above, the approximate dimensions for these cell aggregates is 500 micrometers, as the bar in the photo measures 250 micrometers. It seems that this is the upper limit, for now, for the site of these clusters, however as the technology is already developed, printing larger cell clusters will come in time.

Near future applications of this process would include tissue and organ printing for organ donations and replacements. Furthermore, as hESCs are more commonly being stored at birth as a potential future resource for medical advances, the likelihood of the host body rejecting these tissue implants will severely decrease as the body will recognize them as their own. Having mass produced tissue that reacts the same way the human body does also leads to many other options for testing. Instead of risking lives with a drug that is previously untested in humans, scientists would be able to perform simulated in-vivo testing on life like samples. Additionally, with the innumerable amount of new drugs and products coming into the market every year, the ability to perform toxicity tests on specific types of cells or organs is remarkable. These are only a few of the possibilities that the combination of three dimensional printing and tissue engineering can produce, with new and amazing leaps being taken every day.

As tissue engineering creates a stronger bond between biotechnology and mechanics, 3D printing can also be applied to the field of creating prosthetics. Instead of employing active drugs or stem cells, a 3D printer can be manipulated to print a plethora of replacement body parts and can be customized to be as real or unique looking as each patient wants.
 * Section 3: Prosthetics**

One of the easiest pieces to print on a 3D printer is a tooth. As a replacement tooth does not require complex nerve endings and merely needs to stay in place, be comfortable and look natural it is very easy to print and implant a tooth. In a paper by Cross et. al, a prosthetic tooth was printed using a synthetic resin material, shown below in Figure 6, being compared alongside wax model and a cobalt chrome template [18]. This 3D printed model tooth was then implanted in the patient. Below in Figure 7 are photographs before and after the surgery, where it is nearly impossible to tell which teeth are natural and which are a marvel of technology. Because of 3D printing, replacements like this are becoming more inexpensive and common.
 * Figure 6: Comparison of Replacement Teeth [18]**


 * Figure 7: Before and After Dental Photos With 3D Printed Implanted Tooth [18]**

While printing a tooth is a worthy achievement, it pales in comparison to some of the other prosthetics available. Based on the advances of the technology shown above, 3D printing can easily print custom prosthetics for patients who require leg amputations [19]. Although these may be practical, patients often feel embarrassed or incomplete with the shape that these prosthetics provide. Scott Summit, the man behind Bespoke Innovations has found a way to intertwine these medical accommodations with personal flair using what he has termed "fairings." Fairings are "specialized coverings that surround an existing prosthetic leg, accurately recreating the body form through a process that uses three-dimensional scanning to capture the unique leg shape [20]." Summit shows that 3D printing does not only have to be functional but can also be beautiful, with a few examples shown below in Figures 8, 9, and 10.


 * Figure 8: Bespoke Innovations Fairings [20]**


 * Figure 9: Bespoke Innovations Fairings [20]**


 * Figure 10: Bespoke Innovations Fairings [20]**

Most recently, 3D printing has been a major topic in the news surrounding an artificial prosthetic. Last week, scientists in Scotland were able to create a printed pinna, or an external human ear. Dr. Reiffel et. al. have published a report with their findings in PLoS | One [21]. The pinna was designed using CAD technology and was printed into cartridge bored molds. After the molds were allowed to gel, they were implanted onto the dorsal sides of lab rats to facilitate in-vivo testing. Clearly, this research is still in its earlier stages, but the possibilities that testing like this opens are limitless. The versatility that 3D printing allows in printing substance, mechanisms, and its ability to adapt itself into almost every scientific field shows why it is the ultimate wave of the future.

__References__
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 * 2) Sanders, Ian. "Stratasys Advertisement" //Twitter.// 12 February 12 2013. [|Whatever Your Game]
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 * 5) Anonymous. "Bespoke Innovations" Bespoke Innovations. Web. 24 March 2013. []
 * 6) Reiffel, Alyssa. "High-Fidelity Tissue Engineering of Patient-Specific Auricles for Reconstruction of Pediatric Microtia and Other Auricular Deformities"//PLOS | One.// 8.2 (2013). []