Cartilage Tissue Engineering via Cell Aggregation

All experiments involving rats were conducted under the supervision of UCLA DLAM and USC DAR. Prior to implantation, primary pre-chondrocytes from enzymatically digested 5–6 week old human limbs were isolated using FACS and re-aggregated in ultralow attachment plates (Nunc)^{63 (link)}. Fetal cartilage was dissected from 17-week old fetal tissues, enzymatically digested and re-aggregated for up to 48 in vitro. PSC-derived chondrogenic aggregates were generated as described above from a stable line generated in the Evseenko lab expressing GFP^{64 (link)}. Primary and PSC-derived cell aggregates were then implanted sub-cutaneously into 6, 2 month old, athymic male rats (Harlan labs); no randomization nor blinding was used. 5 aggregates (~200,000 cells each) were implanted to each rat. Day 60 PSC-derived aggregates (2 per rat) were also implanted into full thickness - 1 mm in diameter—osteochondral defects generated in the knee joint of 6 athymic rats using the surgical procedure previously described by our group^{65 (link)}. The chondrosphere aggregates were additionally fixed in place using EVICEL® Fibrin Sealant (Ethicon). After 4 weeks tissues were harvested and analyzed using histological techniques.