Simultaneous regeneration of full-thickness cartilage and subchondral bone defects in vivo using a three-dimensional scaffold-free autologous construct derived from high-density bone marrow-derived mesenchymal stem cells

Background: In recent years, several methods have been developed for repairing full-thickness cartilage defects by tissue engineering using mesenchymal stem cells. Most of these use scaffolds to achieve sufficient thickness. However, considering the potential influence of scaffolds on the surrounding microenvironment, as well as immunological issues, it is desirable to develop a scaffold-free technique. In this study, we developed a novel technique, a scaffold-free autologous construct derived from bone marrow-derived mesenchymal stem cells (BM-MSCs), and successfully use this technique to regenerate cartilage and subchondral bone to repair an osteochondral defect in rabbit knees. Methods: BM-MSCs were isolated from bone marrow liquid aspirated from the iliac crest of rabbits. After expansion in culture dishes and re-suspension in 96-well plates, the cells spontaneously aggregated into a spheroid-like structure. The spheroids were loaded into a tube-shaped Teflon mold with a 5-mm height and maintained under air-liquid interface conditions. These loaded spheroids fused with each other, resulting in a cylinder-shaped construct made of fused cells that conformed to the inner shape of the mold. The construct was implanted into an osteochondral defect in rabbit knees and histologically analyzed 24 and 52 weeks after implantation using Wakitani's scoring system. Results: Both bone and cartilage were regenerated, maintaining a constant thickness of cartilage. The mean histological score was 10 +/- 1.7 in the 24-week group and 9.7 +/- 0.6 in the 52-week group. There was no significant difference between the 24- and 52-week groups in either parameter of the score, indicating that no deterioration of the repaired tissue occurred during the intervening period. Conclusions: Using our novel technique, which employs a three-dimensional scaffold-free autologous construct derived from BM-MSCs, we successfully achieved simultaneous regeneration of bone and cartilage for up to 1 year in vivo. This method has potential for clinical use as a safe and effective method for repairing bone and cartilage defects.