1. 3D PRINTING SKIN GRAFTS FOR FACIAL RECONSTRUCTION Background Thousands of individuals worldwide are in need of a skin tissue transplant, whether due to large burns, wounds, ulcers, infections, or for repair prior to surgery. Skin transplantation surgery is the removal and relocation of healthy, fully-functioning skin from one area of the body to an area that is severely damaged. There are several different types of skin grafts by which this transplantation can be completed. The most accessible, though also the least sufficient, is an autograft. This is the use of the patient’s own skin, commonly harvested from the inner thigh, buttocks, below the collar bone, around the ear, and the upper arm. However, these are often insufficient and an alternative method for skin grafting is necessary. Currently, doctors are working alongside biomedical engineers to develop the innovation of three-dimensionally printing this synthetic skin. Doctors take already present, healthy tissue, and even stem cells extracted from the patient, and use them to harvest more of those cells, which could then be inputted into the 3D printer. The printer in turn prints the desired structure. If successful, this breakthrough has the potential to be far more cost and time efficient, eliminating the need for a transplant waiting list. This would mean patients could get the immediate medical attention they need and be ensured the proper, personalized skin tissue their body requires for a healthy transplant each and every time. Anatomy of the Skin To adequately understand the process of 3D bioprinting skin grafts, one must first understand the anatomy of the skin and its functions. The skin is not only the largest organ in the human body, but it is also one of the most vital. It consists of various specialized cells and structures that develop together to serve as a protective barrier to the human body. The epidermis is the outer layer of the skin. It is comprised of five sub-layers: the stratum basale, stratum spinosum, stratum granulosum, stratum licidum and the stratum corneum. The stratum basale is the bottom sub-layer that pushes newly formed cells into the higher sub- layers. These cells eventually go through each sub-layer and reach the top sub-layer known as the stratum corneum. Here, the now-dead skin cells are shed every two weeks, and replaced by new skin cells. The next significant layer of the skin is the dermis; this is seen in the large middle section of the image below. This is made up of three different types of tissue: collagen, elastic tissue and reticular fibers. There are two sub-layers in the dermis. The first is referred to as the upper papillary, which is a slim arrangement of collagen fibers. The second, made up of thick collagen fibers, is known as the lower reticular layer. The dermis serves many purposes for the human. In this section, hair follicles and oil glands are located. The subcutaneous layer, also known as the hypodermis, is the bottom third layer of the skin. This layer consists of fat and connective tissues, and it houses large blood vessels and nerves. The hypodermis regulates the temperature of the skin and the human body. It is also the region where irregularities such as rashes and abnormal sensations arise. PAST PRESENTFUTURE Three-dimensional (3D) printing is the method of producing 3D objects from a digital image. Though the first documented 3D model was created in 1982 by Hideo Kodama of Nagoya Municipal Industrial Research Institute, it was not until 1986 that Charles W. Hull patented the first working 3D printer. This printer was designed to print objects using his self-proposed method of printing, which he labeled stereolithography. This process is analogous to the form of artwork known as lithography in which one prints on a flat surface that has been treated with a chemical to repel ink in specified areas, allowing the ink to print in all other locations. The printer utilizes ultraviolet (UV) radiation to cure, or solidify, a fluid medium capable of altering its physical state. By exposing specific areas to the UV light, the printer will construct a 3D object by successive laminar buildup, the thin layering of organic tissue or other materials. Hull’s finalized design can be seen in the figure below. A vessel (21) contains a curable liquid (22), or a liquid that is able to be hardened into a solid mold. A UV light is attached to a programmable source (26) that in turn emits a spot of light (27) onto the plane on the surface (23) of the liquid. The position of this spot of light can be digitally controlled by a computer (28). Also attached to and controlled by this computer is a mobile platform (29) that can be translated up or down selectively. After curing one layer of the model-to-be, the platform with the solid material initially formed slides down below the surface, more of the miscible liquid flows across the gap, and the UV light can begin curing the next layer of liquid which will then adhere to the layer below it. Gradually a three-dimensional object (30) is developed by means of this stepwise buildup of integrated laminae (30a), (30b), and (30c) [10]. The process performed by Hull’s original 3D printer can be applied to the direct printing of sacrificial resin molds for the formation of 3D scaffolds from bio materials. These frameworks can then be used for implantation with or without the inclusion of seeded cells. Due to further advancements, the technology now has the ability to directly print tissue. 3D printing tissue is based on three primary approaches; a combination of these approaches are all utilized within bioprinters. The process that this technology performs is a stepwise function, similar to that of Hull’s printer. 4 STEPS IN THE BIOPRINTING PROCESS: 1.Creating a Computer model: These models are made by first taking a digital image either by computed tomography (CT) or magnetic resonance imaging (MRI) scans. These images will provide the printer with the detailed structure and configuration of the tissue on a cellular level that it needs to identically reproduce it. 2.Selecting a printer: There are three primary types of printers. Standard bioprinter (NovoGen MMX)- This bioprinter contains two separate robotic heads, one that assists in placing down the human cells, and another that places water-based layers down to hold together the tissues. Six-axis printer- This type of printer is presently being used for research at the University of Louisville’s Cardiovascular Innovation Institute. While most printers build materials from bottom to top in a layer-by-layer process, these printers can begin at any layer and expand from there. Inkjet bioprinter- This type of bioprinter is currently being used for research at Wake Forest University, and is greatly beneficial when bioprinting skin because it allows for the use of a variety of cell types to be placed directly into the ink cartridge. 3.Creating the ink: A layer-by-layer process, similar to that of the actual bioprinting process, is necessary when creating the “bio-ink” used in inkjet printers. The computer image is used first in this procedure to create a design for the figuration of the tissues needed for the patient. Cells are then layered strategically between water-based coatings such as collagen or hydrogel. 4.Stepwise laminar bioprinting process: This type of printing uses thermal mechanisms to eject drops of ink onto the specific chemical being used. These drops solidify, leading to the formation of the desired structure in a layer-by-layer process. In regard to 3D bioprinting, the amount of potential advances to be made is vast. There are several proposed ideas for further development: Mass generation of mini “tissue blocks” Printing functionally adaptive material Developing a bioreactor Minimally invasive robotic surgical tool, capable of printing in vivo Evidently, 3D printing has not only come a long way, but has further impending successes ahead of it. With dedicated bioengineers working alongside doctors, these medical advancements are not far into the future. Outside of the aforementioned advancements in transplantation surgeries, 3D printing has the potential to lead to new drug discovery, analyses of chemical, biological, and toxicological agents, and research. These possibilities, coupled with already present capabilities of printing functional facial skin grafts and the promise for sustainability, put 3D bioprinting at the forefront of medical innovation. MATERIALSSUSTAINABILITYEFFICINCY CURRENT SKIN GRAFTS Autograft: skin removed from area on patient referred to as “donor site” Allograft: skin donated from another individual as transplant The current process for transplanting skin is lengthy, time- consuming, and requires a donor waiting list. In creating these grafts, excess skin is taken from the donor site and then cut to fit the patient’s wound, resulting in wasted resources. 3D PRINTED SKIN GRAFTS Water-based coating: support for cells composed of either collagen or hydrogel Somatic cells: body cells inputted into the printer head. Materials are readily available, due to the skin cell’s involuntary process of mitosis. Computer scans and imaging, detailing the wound, allow for a more precise and accurate graft that can be printed on location. CAITLIN LUCAS & MADELINE SPIEGEL UNITED STATES ARMY RESEARCH The United States Army is currently conducting research due to the progress and success these bioprinters have made in the medical field with their ability to create artificial skin grafts. The work of these bioprinters has the potential to significantly improve the lives of thousands of American veterans. The Armed Forces Institute of Regenerative Medicine (AFIRM) led by the Wake Forest Institute for Regenerative Medicine (WFIRM) is presently receiving approximately $50 million in funds from the United States Army and the Department of Defense. These funds will be used to facilitate research on 3D bioprinters and their ability to produce skin for wounded warriors.