Immunohistochemistry and Ultrastructural Evaluation of the Interaction Between Nano-Hydroxyapatite/β-Tricalcium Phosphate Composite Spheroids and Bone Marrow-Derived Mesenchymal Stem Cells in a 3D Cell Culture Model
Igor Da Silva Brum, Carlos Nelson Elias, Lucio Frigo, Bianca Torres Ciambarella, Debora Ornelas, Simone Carvalho, Erika Cortez, Alessandra Thole, Ana Lúcia Rosa Nascimento, Karina Ribeiro Silva, Ivonete Sena Dos Santos, Jorge José De CarvalhoThe nano-hydroxyapatite/β-tricalcium phosphate composite (nano-HA/β-TCP) is widely used in various medical and dental procedures. The absence of in vivo toxicity of nano-HA/β-TCP has been extensively studied, and it is considered one of the most effective synthetic biomaterials for promoting cell differentiation in bone regeneration. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are the primary cell type involved in the osteoinductive process of guided bone regeneration following injury. In the present study, rat BM-MSCs were cultured with nano-HA/β-TCP (80/20%) composite spheroids, and the interaction between the cells and the composite was analyzed using transmission electron microscopy (TEM). Ultrathin sections examined by TEM showed extensive interaction between nano-HA/β-TCP and BM-MSCs. Semi-thin sections stained with toluidine blue revealed the incorporation of the biomaterial into the cell cytoplasm. For the immunohistochemistry analysis, eight adult male Wistar rats weighing approximately 300 g were used in each group. Two bilateral, non-critical-sized 3 mm defects were created in the parietal bones of the calvaria: Control, Bio-Oss®, and Blue Bone® (n = 24) during a 12-week experimental period. Bone formation was evaluated through osteonectin and osteopontin expression. At the ultrastructural level, internalization of the biomaterial and close association with the endoplasmic reticulum (ER) and mitochondria were observed. TEM analysis also revealed no harmful effects on the cells, such as apoptotic or necrotic bodies or cell lysis. These findings indicate that the nano-HA/β-TCP composite demonstrates in vitro biocompatibility and interacts appropriately with BM-MSCs, including incorporation into the cell cytoplasm. In vivo, the Blue Bone® group exhibited superior bone formation when compared with the other groups.