BackgroundIn order to enhance cartilage regeneration, surface modification of the cubic micro-cartilage with the collagenase treatment was tested and its efficacy to tissue engineer ear cartilage was investigated. Materials and methodsHarvested cubic micro-cartilages were treated with collagenase with different digestion time (0, 15, 60, and 120 min). Histological, ultrastructural (SEM and TEM), and Western blot analyses were carried out. Subsequently, A total of 45 dogs were used to tissue engineer ear cartilage. Using collagenase-treated micro-cartilage, the ear cartilage regeneration with the prepared dilution (8, 12.5, 25, 50, 100%) of micro-cartilage block seeding was performed to determine the minimum amount of cartilage tissue required for ear tissue-engineering (n = 6 at each point in each group). At 10 weeks after surgery, samples were resected and subjected to histochemical and immune-histological evaluation for cartilage regeneration. ResultsIn vitro study on micro-cartilage morphology and western blot analysis showed that collagenase digestion was optimal at 60 min for cartilage regeneration. In vivo evaluation on the reduced proportions of micro-cartilage block seeding onto implant scaffolds under 60-min collagenase digestion determined the minimum amount of cartilage tissue necessary to initiate a one-step ear cartilage regeneration in a canine autologous model, which was 12.5–25% of the original ear size. ConclusionTissue-engineering ear cartilage from limited volume of donor cartilage can possibly be achieved by the collagenase treatment on micro-cartilage to expand cartilage regeneration capacity, application of cytokine sustained-release system, and seeding on a suitable ear scaffold material.