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Whole-Organ Tissue Engineering Kidneys: Kidneys, Harvested from Pigs, Saving Human Lives

D. Ezra Aurian-Blajeni Engineering 110 Professor Forbes

The role of the kidney in the human body is crucial to survival. The kidney, in short, is a complex filtration unit that, in one day, can process about two hundred quarts of blood to remove about two quarts of waste products and excess water that leave the body in the form of urine.[1] In addition, the kidney is an important regulator of blood pH, concentrations of several electrolytes, and even red blood cell production.[3] Unfortunately, chronic kidney disease (CKD) is not uncommon. As of 2010, more than ten percent of adults in the United States, aged twenty and older, have some form of CKD.[3] Often, the progression of CKD can be hindered by measures, such as dietary restrictions, but it can sometimes lead to end-stage renal disease (ESRD).[1] ESRD is the complete, or almost complete, loss of kidney function, caused primarily by diabetes and/or hypertension (high blood pressure).[1] There are only two treatments for ESRD: dialysis and whole organ transplantation. Dialysis (the artificial filtration of the bloodstream), can be expensive, resource intensive, and even risky; dialysis procedures may be required up to four times per day, and the risk of clotting and/or infection of a vascular access point is a constant reality for dialysis patients.[2][3] Whole organ transplantation, on the other hand, is the only treatment of ESRD that restores full function of the kidney to the patient.[5] From 2006 to 2009, the annual cost per patient has been less than half of the cost for the least expensive dialysis treatment.[2] Additionally, there is no need to regularly access a patients bloodstream, eliminating the need for a vascular access point and precluding the clotting or infection thereof. The major barrier to whole organ transplantation is the availability of transplant donors. In February 2011, the waiting list for kidney recipients was almost 90,000.[4] In 2010, about 16,000 ESRD patients received kidney transplants, making it the most commonly transplanted organ in the United States.[4] It is obvious that the demand for donor kidneys far outpaces the supply.

However, whole organ transplantation is not without its own risks. When a patient receives an organ transplant, the bodys immune system recognizes the donors organ as a foreign entity and will attack it as if it were a giant mass of invasive bacteria. As a result, a transplant patient is prescribed a regimen of immunosuppressant medications, which, in most cases, must be adhered to for the rest of their lives, or else the patient risks the bodys rejection of the transplanted organ.[4] These immunosuppressants indiscriminately suppress the entirety of the patients immune system. Consequently, the patient is put at risk for different kinds of infections, cancer, and, if taken for long enough, even kidney damage, hypertension, and diabetes.[4] In effect, a kidney transplant patient might require another kidney transplant after a relatively short amount of time. But what if there was a way to fabricate a personalized kidney, ex vivo (outside the body), using a patients own stem cells? Would this not both eliminate the risk of transplantation rejection, as well as preclude the need for a donor organ to become available?

Whole-organ tissue engineering has become an area of great interest in the past several years. Currently, there are no good solutions for complex, solid organs, like the kidney or liver. It has been clinically been demonstrated, however, that artificially engineered tissue can partially or completely replace a patients damaged tissue with great success. Among many, one notable example involves the complete replacement of a section of an airway in a human patient.[6] A number of groups have taken some encouraging first steps in the direction of engineering whole, complex, solid organs. While there has been much excitement over this initial progress, no definitive functional solutions have been demonstrated yet, not even in animal models. There is still much work to be done.

In 2008, Macchiarini et al.[6] published the results of a less than two-year study on the effectiveness of a novel type of airway transplant. A portion of a patients left main bronchus (part of the airway near the lung) was removed and replaced with a segment of tissue-engineered trachea. Omitting many details, the engineered tissue was produced as follows. First, a portion of trachea from a deceased donor was decellularized. That is, all the donors cells were washed out of the tissue with detergents, leaving only the extracellular matrix (ECM). Previously prepared samples of the patients own mesenchymal stem cells, obtained from the patients own bone marrow, were, via biochemical manipulation (involving cell signaling molecules), directed to differentiate into autologous (the patients own) chondrocytes (cartilage-producing cells). These chondrocytes were seeded (applied) to the outside of the tracheal ECM. Similarly, the patients own epithelial cells, obtained from a sample of a section of the patients own (healthy) bronchus, were cultured (grown) and applied to the lumen (inside) of the tracheal ECM. The seeded ECM was then put into a seemingly unsophisticated bioreactor (a closed chamber that must closely mimic a tissues physiological conditions in order for the engineered tissue to develop correctly). The bioreactors rotating design was simple, yet elegant, involving only autoclavable (able to be sterilized) structural elements, a tiny motor, and two types of culture media (liquid solutions that contain nutrients, growth factors, and other biomolecules that promote cell growth and division), one for the lumen and the other for the chondrocytes, and closely resembled the liquid and air-exposed environments to which a human trachea is exposed. In only four days, the engineered trachea was prepared to be grafted.[6] The transplant operation was successful. Four days after the operation, the transplanted tissue was nearly indistinguishable from the patients own tissue nearby. The patient was released only ten days after the operation. Within two months, the patients lung function

improved dramatically. Perhaps the most notable aspect of this procedure, aside from the incredibly quick recuperation, is the fact that the patient never had to take any form of immunosuppressant medication. The basic protocols utilized in this procedure are the basis for modern tissue engineering.[7] First, a donor organ is decellularized to reveal an ECM. Then, it is seeded with appropriate stem cell types, derived from the patients own tissues. Next, the seeded ECM is put into a specialized bioreactor for a period of time and constantly monitored. Finally, the artificial tissue is implanted into the patient. This general format for fabrication and implantation of an artificial tissue has enjoyed much success and is widely used.[7] Other successful human transplant procedures involving engineered tissues have been clinically demonstrated, including skin and urinary bladder operations.[7] It must be noted that, in the latter two procedures, the engineered tissues were not constructed with human ECM, as in the tracheal tissue described above. These artificially engineered tissues were constructed with ECM from pig tissues.[7] How is this possible? It turns out that proteins in the ECM are highly conserved (that is, they have stayed almost the same throughout evolution), even across very different species.[8] As a result, the proteins in humans ECMs are very much similar to the proteins in pigs ECMs. In addition, the breakdown of the ECM releases cell signals that are specific to the tissue from which the ECM was derived.[8] That is, stem cells that come into contact with kidney ECM that has broken down a little bit will be directed to differentiate into kidney cells. For these and other reasons (logistical simplicity, prevention of spreading human viruses, size similarity), decellularized porcine (pig) organs are ideal candidates for the engineering of some tissues,[8] including kidneys.

There have been a number of publications on porcine kidney decellularization. However, the focus of the study being conducted differs from paper to paper. For example, one publication elaborates on the patency and preservation of the minutiae of the three-dimensional structure of the kidney ECM after decellularization,[9] while another focuses on the reproducibility and efficiency of the decellularization process.[5] It has been proven that human stem cells can differentiate on porcine ECMs. However, there are a number of challenges that must still be overcome. It has not been determined what the best sources of stem cells for the seeding of the ECM are.[8] Neither has an appropriate bioreactor been designed for the seeded kidney ECM,[8] presumably because its physiological conditions are so very different from that of other organs. Another challenge is seeding the endothelium of the kidney ECM.[8] Yet another is how to address the need of an immediate blood supply to the vast and complicated structure of the kidney.[8] In time, these things will be addressed and the dream of an artificial kidney will be attained.

References: 1. The National Kidney and Urologic Diseases Information Clearinghouse. 2009. The Kidneys and How They Work Page - National Kidney and Urologic Diseases Information Clearinghouse. [Online document] http://kidney.niddk.nih.gov/kudiseases/pubs/yourkidneys/ (Visited October 25, 2012) 2. The National Kidney and Urologic Diseases Information Clearinghouse. 2009. The Kidneys and How They Work Page - National Kidney and Urologic Diseases Information Clearinghouse. [Online document] http://www.kidney.niddk.nih.gov/KUDiseases/pubs/kustats/index.aspx (Visited October 25, 2012) 3. Centers for Disease Control and Prevention. 2010. CDC - National Chronic Kidney Disease Fact Sheet 2010 - Factsheets - Publication - Diabetes DDT. [Online Document] http://www.cdc.gov/diabetes/pubs/factsheets/kidney.htm (Visited October 25, 2012) 4. NIH News in Health. 2011. Organ Donation - NIH News in Health, March 2011. [Online document] http://newsinhealth.nih.gov/issue/Mar2011/Feature1 (Visited October 25, 2012) 5. Sullivan DC, Mirmalek-Sani SH, Deegan DB, Baptista PM, Aboushwareb T, Atala A, Yoo JJ. 2012. Decellularization methods of porcine kidneys for whole organ engineering using a high-throughput system. Biomaterials. 33:775664 6. Macchiarini P, Jungebluth P, Go T, Asnaghi MA, Rees LE, Cogan TA, Dodson A, Martorell J, Bellini S, Parnigotto PP, Dickinson SC, Hollander AP, Mantero S, Conconi MT, Birchall MA. 2008. Clinical transplantation of a tissue-engineered airway. Lancet. 372:202330 7. Badylak SF, Weiss DJ, Caplan A, Macchiarini P. 2012. Engineered whole organs and complex tissues. Lancet. 379:94352 8. Badylak SF, Taylor D, Uygun K. 2011. Whole-Organ Tissue Engineering: Decellularization and Recellularization of Three-Dimensional Matrix Scaffolds. Annual Review of Biomedical Engineering. 13:2753 9. Orlando G, Farney AC, Iskandar SS, Mirmalek-Sani SH, Sullivan DC, Moran E, AbouShwareb T, De Coppi P, Wood KJ, Stratta RJ, Atala A, Yoo JJ, Soker S. 2012. Production and implantation of renal extracellular matrix scaffolds from porcine kidneys as a platform for renal bioengineering investigations. Annals of Surgery. 256:363-70