Time Passages: Defining the Limits of Human Auricular Chondrocyte Usability for Tissue Engineering
Jaime L. Bernstein, BS1, Benjamin P. Cohen, MS2, Yoshiko Toyoda, BA1, John P. Morgan, PhD1, Alice Harper, BA1, Lawrence J. Bonassar, PhD2, Jason A. Spector, MD1.
1Weill Cornell Medical College, New York, NY, USA, 2Cornell University, Ithaca, NY, USA.
BACKGROUND: Current autologous reconstructive options for microtia, as well as other auricular deformities secondary to trauma and oncologic resection, have significant shortcomings, which has sparked an increasing interest in cartilage engineering. Previously, using bovine auricular chondrocytes, we fabricated high fidelity patient specific auricular scaffolds, which displayed long term stability following implantation as well as structural, mechanical, and biochemical properties indistinguishable from that of native bovine auricular cartilage. However, clinical translation of these scaffolds mandates the use of approximately 250 million human auricular chondrocyte (hAuCs) per construct while a clinically obtainable amount of auricular tissue (i.e. 1 gram from the contralateral conchal bowl and/or microtic remnant) only yields approximately 10 million cells. It is known that repeated passaging of chondrocytes to expand cell number leads to de-differentiation and loss of their chondrogenic potential. However, the few studies available in the literature assert that human auricular chondrocytes can be passaged no more than 2-3 times prior to loss of chondrogenic capacity. We sought to specifically determine the number of passages that both maximizes the cellular expansion while minimizing dedifferentiation.
METHODS: Human auricular chondrocytes (HAuCs) were isolated from separate otoplasty specimens. The HAuCs were expanded and cells from passage 3, 4, and 5 were encapsulated separately into type I collagen 8mm diameter disc hydrogels with a cell density of 25 million cells/mL. The constructs were then implanted subcutaneously in the dorsa of nude mice, and harvested after 1 and 3 months for analysis.
RESULTS: Constructs containing passage 3, 4, and 5 chondrocytes all maintained cylindrical geometry. After 3 months, passage 3 and 4 discs on average contracted 25.00% and 26.17%, respectively. Interestingly, passage 5 discs on average contracted only 17.96%, which was significantly less (p<0.05). Regardless of the passage number, all constructs developed a white cartilage like appearance and had flexibility similar to native human auricular cartilage. Histologically, the cartilage produced from passage 3, 4, and 5 resembled native auricular cartilage with an organized perichondrium composed of collagen, cellular lacunae, a rich proteoglycan matrix, and a dense elastin fiber network evident by safranin-O and Verhoeff's stains respectively. Biochemical and mechanical analyses are pending.
CONCLUSIONS: Contrary to prior reports, we have shown that human auricular chondrocytes can be expanded up to passage 5 with no apparent difference in the chondrogenic capacity. In fact, passage 5 constructs experienced significantly less contraction that passage 3 and 4, while still producing cartilage. These data indicate, for the first time, that later passaged human auricular chondrocytes have the same potential to be used for cartilage engineering as earlier passaged cells. Understanding the chondrogenic capacity of later passaged chondrocytes is crucial for the successful translation of human auricular tissue engineering, as more passages may allow for expansion of patient specific donor chondrocytes to the requisite number needed for construct fabrication.
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