Bio-3D printing of scaffold-free osteogenic and chondrogenic constructs using rat adipose-derived stromal cells

Background: Although autogenous bone implantation is considered to be the gold standard for the reconstruction of bone defects, this approach remains challenging when treating extensive bone defects (EBDs). Therefore, artificial materials (AMs) such as artificial bone and scaffolds are often used for treating EBDs. Nevertheless, complications such as material failure, foreign body reaction, and infection are common. To overcome these issues, we aimed to develop a new treatment for an EBD using scaffold-free adipose-derived stromal cells (ADSCs) to fabricate chondrogenic/osteogenic-induced constructs without AMs. Methods: ADSCs were obtained from the subcutaneous adipose tissue of 8-week-old female Wistar rats (n = 3) and assessed to determine their potential for multilineage differentiation into adipocytes (Oil Red O staining), chondrocytes (hematoxylin and eosin, Alcian blue, and Safranin O staining), and osteoblasts (Alizarin red and von Kossa staining). Spheroids (n = 320), each containing 3.0 x 104 ADSCs, were then used to fabricate scaffold-free cell constructs using a bio-3D printer with a needle array. The spheroids and constructs were stimulated with induction medium to induce chondrogenic and osteogenic differentiation. The induced cartilage- and bone-like constructs were finally evaluated using micro-computed tomography (ACT) and histological analysis. Results: The collected ADSCs were capable of trilineage differentiation, and were successfully used to produce scaffold-free constructs. The fabricated constructs (n = 3) exhibited equivalent strength (load, 195.3 +/- 6.1 mN; strength, 39.1 +/- 1.2 kPa; and stiffness, 0.09 +/- 0.01 N/mm) to that of soft tissues such as the muscles in the uninduced condition. In chondrogenic induction experiments, Alcian blue and Safranin O staining confirmed the differentiation of the constructs into cartilage, and cartilage tissue-like structures were produced. In the osteogenic induction experiment, Alizarin Red and von Kossa staining showed calcium salt deposition, and ACT images confirmed the same calcification level as that of the cortical bone. Conclusions: Scaffold-free constructs consisting of ADSCs without an AM were fabricated, and cartilage- and bone-like tissues were successfully generated, demonstrating their potential for bone reconstruction.