Title:
Summary
Chronic Kidney Disease is a debilitating disease that affects more than 26 million people in America. Currently, the only treatments available for those with CKD is kidney dialysis, a costly and time-consuming treatment, or a kidney transplant. Acquiring a suitable kidney for transplantation can take several years and there is a chance of rejection of the kidney by the bodyʻs immune system. Researchers are currently working towards developing technology that would allow scientists to build kidneys and other organs using a 3-D printer that integrates cell samples from the patient. This would eliminate the transplant waiting list and the chance of rejection. However, current technology is still far from that goal. A major setback is designing a way to vascularize printed tissue that would function similarly to biological tissues. Currently, researchers are able to construct functional vascular systems in micro settings, but have been unable to take the technology to a larger scale because of complexity in the number of vessels and the varying sizes that can be present in a small area of tissue. INSERT APPROACH SUMMARY
Significance
If kidneys become damaged, they may not function properly and the individual could develop Chronic Kidney Disease (CKD), a disease that affects over 26 million Americans (nih.gov). Patients with CKD can experience debilitating symptoms such as anemia, weak bones, poor nutritional health, nerve damage, and high blood pressure (nih.gov). When CKD progresses, it can develop into kidney failure, which requires the patient to undergo dialysis or a kidney transplant to stay alive (nih.gov). Currently, there are over 93,000 people waiting on the kidney transplant list, with an average wait time of five to ten years (kidneyhi.org). Kidney dialysis, while effective, does not cure the patient and is a time-consuming treatment that patients have to undergo for the rest of their lives if they do not receive a new kidney (kidneyhi.org). A kidney transplant, on average, doubles the patientʻs life expectancy (kidneyhi.org). Due to an increase of patients with kidney disease, it is expected that the number of people waiting for a kidney could exceed 100,000 people and the wait time would increase to over 10 years (kidneyhi.org). The longer that a patient is on dialysis, the lower the success rate for a kidney transplant and increase the chance of complications from the surgery (kidneyhi.org).
Background
Kidneys are responsible for removing waste and excess water from the body, as well as maintaining a healthy balance of water, salts, and minerals (nih.gov). A complex vascular system within the kidney transports the blood through the filtration process. Those with CKD are unable to maintain homeostasis without dialysis or a kidney transplant. However, a new technology has the potential to change that. 3-D bioprinting is a printing technology that has the potential to revolutionize many areas of science and medicine (Murphy and Atala, 2014). This technology entails using a specialized printer that carefully lays down thin layers of bioinks made of human cells to form complex, living structures (Murphy and Atala, 2014). 3-D bioprinting can be applied in a myriad of areas. With 3-D bioprinting, a kidney could be grown from a patientʻs own cells, eliminating the wait time to receive a transplant and decreasing the risk that the patientʻs body rejects the kidney (Murphy and Atala, 2014).
Current 3-D printing technology is unable to construct functional kidney tissue because inducing vascularization in printed tissue has so far been unsuccessful (Zhu, et al). In tissues, growth factors such as the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) naturally stimulate the growth of blood vessels. These growth factors then activate receptors on endothelial cells in the lining of pre-existing blood vessels. The activated endothelial cells secrete enzymes called proteases that break down the outer layer of the blood vessel and the endothelial cells escape into the surrounding matrix and proliferate and join together, forming connections to adjacent vessels. However, this process takes one week to develop a mm of a blood vessel (Adair). Printing tissue and then vascularizing it by stimulating natural vascular growth leaves the tissue in a hypoxic state until the vessels have fully developed through the entire tissue, by which time the tissue would have died from lack of oxygen and necessary nutrients. (Murphy and Atala, 2014). A new method includes printing pre-vascularized tissue that can then be inserted into a host (Zhu, et al). Researchers showed that grafting pre-vascularized 3-D printed tissue into mice skin successfully joined with the mouse blood vessel network and showed blood circulation through the printed vessels (Zhu, et al).
Using 3-D printing to construct blood vessels is done using various methods. One such method uses microscale continuous optical bioprinting, which essentially uses light to focus the printer to develop smaller and more complex structures out of hydrogel (Zhu, et al). The hydrogel is macromolecular polymer gel constructed of a network of crosslinked polymer chains that functions as a substrate for injected cells that mimic the natural vascular environment. This method is only able to print structures the size of a pencil eraser with a thickness of about 0.6 mm, making it difficult to use for larger, complex tissues without being able to do it on a larger scale (Zhu, et al).
Overall goal: Improve 3-D printed vascular systems by constructing a large-scale functional blood vessel system
Hypothesis: Using a split-cell approach will allow larger areas of functional vascular systems to be constructed in a shorter amount of time than normal angiogenesis
Aims:
To demonstrate that separated blood vessels can be connected by inserting angiogenesis growth factors (AGF)
To demonstrate that a sheet of vascularized tissue can be constructed by connecting micro sections of blood vessels imbued with AGF
Rationale:
Current blood vessel printing technology allows scientists to print thin blood vessel systems about the size of a pencil eraser that can join and function with vascular systems in mice skin, but so far they are unable to print more complex systems that would be able to vascularize entire organs or other thick tissues. Constructing a sheet of vascularized tissue would be an important step towards building an entire 3-D printed organ because it would allow larger areas of functional tissue to be developed. Using the 3-D bioprinting method that uses microscale continuous optical bioprinting will work for this experiment because it has been demonstrated that blood vessels constructed that way are functional, just on a microscale and this approach uses similar procedure except it constructs micro versions of blood vessels that will be pieced together to form a larger one.
Approach:
Using a 3-D computer model of a vascular network, sections will be divided into the entire system to form sections about one mm by one mm with a thickness of 0.6 mm. Using the microscale continuous optical bioprinting, hydrogel scaffolds will be constructed for each section with umbilical vein endothelial cells acquired from ATCC will be injected. Printed structures will be kept in endothelial cell growth medium (EGM-2, Lonza) in a compartmented base. After seven days, the compartmentations will be removed and VEGF and FGF will be added to each section to stimulate angiogenesis and connect the sections together. After another seven days, a Cell viability assay (LIVE/DEAD® Viability/Cytotoxicity Kit, Invitrogen) will be used to measure the survivability of the printed blood vessels.
Feasibility:
Innovation:
This method of engineering vascularized tissue is better because instead of constructing large areas of vascularized tissue, which is still not feasible because of the complexity of vascular systems, this method only requires printing tiny sections of blood vessels and then using angiogenesis growth factors (AGF) to connect adjacent vessels together. Printing small sections of blood vessels has been demonstrated to function similar to biological blood vessels in mice and tissues continuously grow new blood vessels every day using AGF. This method pieces the two processes together. No one has done this before because most of the focus has been on innovating methods to construct the organs themselves.