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BACKGROUND:
Reconstruction of external-ear has been one of the most challenging problems in plastic and reconstructive surgery. Currently autologous costal cartilage and synthetic scaffolds (e.g., Medpor) are the most common candidate frameworks. However, neither of them provides fully satisfying results because of inconsistent cosmetic outcome, extrusion, and other surgical complications. We propose that a novel low density collagen scaffold combined with auricular chondrocytes could be a possible alternative for auricular reconstruction. The aims of this study were: 1) to assess the most favorable condition for in vitro pre-culture of scaffold seeded with cells before in vivo implantation; 2) to determine whether auricular chondrocytes seeded on low density collagen scaffold can form neo-cartilage in vivo comparable to native cartilage morphologically, histologically and biochemically.
METHODS:
Commercially available low density collagen scaffold was acquired from Kensey Nash Corporation. Auricular chondrocytes from sheep were isolated and placed in culture for proliferation. 5 million cells suspended in 0.2 ml saline were seeded onto both of sides of low density collagen scaffold (5 mm diameter and 2 mm depth). The constructs were pre-cultured in static and dynamic (Rotational Oxygen Permeable Bioreactor System (ROBS) and Obital Mixer (Rotomix)) systems separately for 2 days and 2 weeks. After that, all of the constructs were implanted into subcutaneous pockets of nude mice for 6 weeks. The specimens were evaluated histologically, immunohistochemically, and biochemically.
RESULTS:
All of the harvested specimens resembled native cartilage in color, luster and texture (Fig 1). Specimens pre-cultured in dynamic systems (robs, A-D; rotomix, E-H) showed more contiguous cartilage matrix histologically (H&E; Fig 1, A&F). All constructs showed glycosaminoglycan production as evidenced in specimens stained with safranin-O (Fig 1, B&G). Intense immunostaining for type II collagen (Fig 1, D&I), which is consistent with native cartilage, was observed. Very little type I collagen (Fig 1, C&H) was observed. All the specimens showed more total DNA content and glycosaminoglycan than native cartilage controls because indicating hypercellular constructs. The collagen content of the neo-cartilage was comparable to that in native cartilage (Fig 2). The amount of glycosaminoglycan was elevated in all seeded scaffolds, especially those specimens pre-cultured in dynamic systems.
CONCLUSIONS:
This novel low density collagen scaffold seeded with auricular chondrocytes permits contiguous neo-cartilage matrix formation in vivo comparable to native cartilage morphologically, histologically, and biochemically. Pre-culture in dynamic systems improves neo-cartilage formation histologically and biochemcially. This novel scaffold could be an ideal alternative for total external-ear reconstruction. Additional studies in mice and large animal have been performed to determine the feasibility of ear reconstruction using this material as the auricular framework (Fig 3).