Optimizing Human Elastic Cartilage Engineering Using Mesenchymal Stem Cell Chaperones

INTRODUCTION: Current options for autologous reconstruction of pediatric microtia and other auricular deformities secondary to trauma and oncologic resection have significant shortcomings, including suboptimal aesthetic outcomes and morbidity at the costal cartilage donor site. In previous studies, we fabricated high-fidelity human-shaped auricular scaffolds using bovine auricular chondrocytes, which have displayed long-term stability following implantation as well as structural and biochemical properties indistinguishable from that of native auricular cartilage. However, clinical translation of this approach mandates the use of approximately 250 million human auricular chondrocyte (hAuCs) for a full-scale ear, and unfortunately autologous tissue donation generates a limited cell yield and subsequent passaging for cellular expansion also presents the problem of chondrocyte dedifferentiation. We hypothesized that co-culture of human mesenchymal stem cells (hMSCs), which hold a chondrogenic potential, with HAuCs would promote the formation of healthy elastic cartilage while allowing for the use of fewer scarce chondrocytes. METHODS: In order to simulate the shape of the auricular helical rim, an external “ridged” scaffold was designed, optimized for 3D-printing with a Makerbot Replicator 5th Generation and printed using polylactic acid (PLA). HAuCs isolated from discarded otoplasty specimens along with hMSCs extracted from human bone marrow were encapsulated into neutralized 1% type I collagen at a total cell density of 25 million per mL using different mixture ratios (HAuCs/hMSCs: 10/90, 25/75, 50/50, and 100/0). This cell-loaded collagen solution was then injected into 3D-printed ridged external scaffolds, cross linked in situ, and implanted subcutaneously in vivo. Samples were harvested for after 3 months. RESULTS: With the presence of an external scaffold, the constructs demonstrated a significant and impressive volume preservation after 3 months at 10/90, 25/75 and 50/50 cell mixture ratios (10/90: 90.41 ± 6.38%, 25/75: 83.29 ± 15.05%, 50/50: 83.5 ± 5.94%, respectively) compared with the 100% HAuCs-loaded group (33.63 ± 5.75% at 3 months) (P < 0.05). After 3 months in vivo, a white cartilage-like appearance of the tissue formed within the cage was noted in all groups, but preservation of topography of the ridged “helical” feature was observed only at (HAuCs/hMSCs) 10/90, 25/75, and 50/50 cell mixture ratios. Histological staining verified the development of mature elastic cartilage within the constructs after 3 months with chondrocytes seen in lacunae within a proteoglycan collagen matrix and surrounded by a neoperichondrial external layer. CONCLUSIONS: Co-implantation of hAuCs and hMSCs in collagen within an external scaffold produced human elastic cartilage more effectively than 100% hAuC alone, even when hAuC comprised less than half of the implanted cell population. Although it is unclear if this more efficient cartilage formation is the result of differentiation of MSC towards a chondrogenic lineage, a chaperone effect of the MSC, or some combination of both remains unknown, and is the subject of ongoing investigation. Given the scarcity of auricular cartilage donor tissue, reducing the relatively large number of chondrocytes required for the generation of de novo constructs is an important step toward the clinical translation of auricular tissue engineering.