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BACKGROUND: Previously, we bioprinted high fidelity patient-specific ears using bovine chondrocytes encapsulated within a type 1 collagen matrix. A major obstacle to the clinical translation of our approach is obtaining the large number (approximately 250 million) of auricular chondrocytes (AC) necessary for the fabrication of a full sized ear scaffold. Because only a few million ACs can be isolated from microtia patient donor sites (the microtic remnant and the contralateral conchal bowl) and in vitro chondrocyte expansion results in de-differentiation and loss of chondrogenic capacity, alternative cell sourcing strategies are necessary. Here we augment the limited chondrocyte supply with mesenchymal stem cells (MSC), which can be obtained reliably in large numbers, expanded significantly and are known to release chondrogenic trophic factors, so as to fabricate elastic cartilage using significantly fewer AC.
METHODS: Bovine AC (BAC) and MSC (BMSC) were encapsulated within 10 mg/ml type I collagen hydrogels in ratios of 100:0, 50:50 and 0:100 BAC:BMSC with a constant cell density of 25 million cells/ml hydrogel. One mm thick collagen sheet gels were fabricated, and thereafter, 8mm diameter discs were extracted using a biopsy punch. Discs were implanted subcutaneously in the dorsa of nude mice, harvested following 1 or 3 month time points and thereafter analyzed for gross morphology, histology and biochemical composition.
RESULTS: Gross analysis of explanted disks revealed that BAC:BMSC co-cultured disks maintained their size and exhibited native cartilage-like elasticity after 1 and 3 months of implantation. H&E staining demonstrated BAC:BMSC disks developed auricular cartilage characteristics, including cellular lacunae and perichondrial layering, and Verhoeff staining revealed co-cultured disks deposited dense elastin fibers. Additionally, biochemical analysis confirmed that BAC:BMSC disks contained significantly more proteoglycan content than either the BAC or BMSC disks after 1 and 3 months. Furthermore, significantly more proteoglycan content was noted after 3 months compared to 1 month in the co-cultured disks.
CONCLUSIONS: BAC:BMSC co-cultured disks maintained their size and shape after 1 and 3 months of implantation, developed auricular cartilage features and deposited critical cartilage molecular components more readily than did BAC or BMSC alone. The in vivo maturation of co-cultured disks using 50% fewer AC into elastic cartilage demonstrates the potential for MSC to supplement the limited pool of AC for tissue engineered auricular reconstruction approaches. Given the success of this approach we are now determining the minimal fraction of BAC required for formation of auricular cartilage. Further, similar trials are underway using human derived AC and MSC, bringing us significantly closer to the fabrication of patient-specific, high fidelity human auricles for the treatment of microtia.