This study in dogs explored the feasibility of using cartilage fragments removed and discarded during routine palliative surgery for osteochondritis dissecans (OCD) as a source of primary chondrocytes for scaffold-free cartilage tissue-engineering. Primary chondrocytes were obtained from three OCD donors and one age-matched healthy articular cartilage (HAC) donor. After monolayer expansion of primary cells, a three-dimensional spherical suspension culture was implemented. Following this stage, cells were seeded at a high density into custom-made agarose molds that allowed for size and shape-specific constructs to be generated via a method of cellular self-assembling in a scaffold-free environment. Fifty-eight neocartilage constructs were tissue-engineered using this methodology. Neocartilage constructs and native cartilage from shoulder joint were subjected to histological, mechanical, and biochemical testing. OCD and HAC chondrocytes-sourced constructs had uniformly flat morphology and histology consistent with cartilage tissue. Constructs sourced from OCD chondrocytes were 1.5-times (32%) stiffer in compression and 1.3 times (23%) stronger in tension than constructs sourced from HAC chondrocytes and only 8.7-times (81%) less stiff in tension than native tissue. Constructs from both cell sources consistently had lower collagen content than native tissue (22.9%/dry weight [DW] for OCD and 4.1%/DW for HAC vs. 51.1%/DW native tissue). To improve the collagen content and mechanical properties of neocartilage, biological and mechanical stimuli, and thyroid hormone (tri-iodothyronine) were applied to the chondrocytes during the self-assembling stage in two separate studies. A 2.6-fold (62%) increase in compressive stiffness was detected with supplementation of biological stimuli alone and 5-fold (81%) increase with combined biological and mechanical stimuli at 20% strain. Application of thyroid hormone improved collagen content (1.7-times, 33%), tensile strength (1.8-times, 43%), and stiffness (1.3-times, 21%) of constructs, relative to untreated controls. Collectively, these data suggest that OCD chondrocytes can serve as a reliable cell source for cartilage tissue-engineering and that canine chondrocytes respond favorably to biological and mechanical stimuli that have been shown effective in chondrocytes from other animal species, including humans.