Autologous Auricular Cartilage Engineering Using Fibrillar Collagen in an Ovine Model
David A. Bichara, MD, Xing Zhao, MD, Libin Zhou, MDS, Katharine M. Kulig, BA, Irina Pomerantseva, MD PhD, Mark A. Randolph, MAS, Erik Bassett, MS, Cathryn A. Sundback, PhD, Joseph P. Vacanti, MD.
Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
Engineering auricular cartilage in immunocompetent species using autologous chondrocytes remains an elusive topic in the field of tissue engineering. Published studies have failed to demonstrate reproducibility, satisfactory neocartilage formation and most importantly, long-term auricular cartilage shape retention and cell viability. We hypothesized that autologous auricular cartilage could be successfully engineered using an FDA-approved fibrillar collagen matrix utilizing a restricted amount of donor tissue. We further analyzed and compared variables including age, ovine breed, pre-cultuiring in vitro time and distinct bioreactor systems, and the effects these factors would have on the newly formed tissue (Table 1).
Seven sheep were used in this study. Two grams of auricular cartilage was harvested, minced, and digested using dilute collagenase. Isolated chondrocytes were cultured until confluent at P1 (14d) to minimize chondrocyte dedifferentiation. After expansion, 50x10^6 cells/ml were statically seeded onto porous fibrillar collagen scaffolds (10 mm diameter and 2 mm thickness). Cell-seeded scaffolds were cultured in vitro for two or six weeks using Rotational Oxygen-Permeable Bioreactor System (ROBS) at 1 rpm or an orbital mixer at 55 rpm (Fig. 1). Autologous serum was substituted for bovine serum 72h prior implantation. Cell-seeded constructs were implanted subcutaneously on the necks of the same sheep (Ti rings were used for identification at time of explant, Fig. 2A) and on the dorsum of nude mice. Explants were subjected to toluidine blue, safranin-O, and collagen type II immunohistochemical staining.
Upon histological examination, tissue engineered cartilage resembled native sheep auricular cartilage using both 3 and 11 month-old sheep tissue. Neocartilage formation and extracellular matrix deposition was identified in all samples confirmed by toluidine blue and safranin O stains (Fig. 2B and 2C). Collagen type II was further identified by means of immunohistochemistry (Fig. 2D) There were no histological differences between a 2 and 6 week in vitro culture period, and both ROBS and an orbital mixer allowed for ECM deposition to occur prior in vivo implantation.
These data represent important milestones in developing an engineered replacement auricle as a clinical therapy. Neocartilage can be successfully engineered using scant amounts of autologous auricular chondrocytes in an immunocompetent animal model.
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