Scaffold-Free Tissue-Engineered Construct–Hydroxyapatite Composites Generated by an Alternate Soaking Process: Potential for Repair of Bone Defects

Abstract

Mesenchymal stem cell (MSC)–based tissue-engineered construct (TEC)–hydroxyapatite (HAp) composites were developed by an alternate soaking process. The TEC derived from cultured synovial MSCs was alternately immersed in varying concentrations of CaCl₂/Tris-HCl and Na₂HPO₄/Tris-HCl buffers, and HAp formation was analyzed by Fourier transform infrared spectroscopy (FT-IR), wide-angle X-ray diffraction, and scanning electron microscopy (SEM). These analyses clearly demonstrated HAp formation in the TEC. Specifically, SEM assessments showed that spherical HAp crystals of ∼1 μm were directly formed on the surfaces of the cells and extracellular matrix (ECM) fibers. Cytotoxicity from exposure to calcium or phosphate buffers of >100 mM concentrations as assessed by LIVE/DEAD staining and total DNA assays was detected, but such cytotoxicity was not detected following exposure to concentrations of <50 mM. The HAp nanocrystals (ca. ∼500 nm) were formed after 20 cycles in 10 mM calcium or phosphate buffers, and cell survival in the composites was confirmed. Moreover, preliminary implantation of TEC-HAp composites derived from rabbit synovial MSCs to rabbit osteochondral defects exhibited accelerated osteoinduction. These composites may be the first example of a hybrid material that consists of ECM, HAp nanocrystals, and living MSCs, and the TEC-HAp composite could be a unique and useful material for bone tissue engineering.