ABSTRACT
The regeneration of whole osteochondral constructs with a physiological structure has been a significant issue, both clinically and academically. In this study, we present a method using rabbit bone marrow stromal cells (BMSCs) cultured on a silk-RADA peptide scaffold in a specially designed two-chambered co-culture well for the generation of multilayered osteochondral constructs in vitro. This specially designed two-chambered well can simultaneously provide osteogenic and chondrogenic stimulation to cells located in different regions of the scaffold. We demonstrated that this co-culture approach could successfully provide specific chemical stimulation to BMSCs located on different layers within a single scaffold, resulting in the formation of multilayered osteochondral constructs containing cartilage-like and subchondral bone-like tissue, as well as the intermediate osteochondral interface. The cells in the intermediate region were found to be hypertrophic chondrocytes, embedded in a calcified extracellular matrix containing glycosaminoglycans and collagen types I, II and X. In conclusion, this study provides a single-step approach that highlights the feasibility of rabbit BMSCs as a single-cell source for multilayered osteochondral construct generation in vitro.
Subject(s)
Bone Marrow Cells/cytology , Chondrogenesis , Coculture Techniques/methods , Mesenchymal Stem Cells/cytology , Peptides/pharmacology , Silk/pharmacology , Animals , Bombyx , Bone Marrow Cells/drug effects , Calcification, Physiologic/drug effects , Cell Shape/drug effects , Chondrogenesis/drug effects , Collagen/metabolism , Compressive Strength/drug effects , Diffusion , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Extracellular Matrix/ultrastructure , Gene Expression Regulation/drug effects , Glycosaminoglycans/metabolism , Mesenchymal Stem Cells/drug effects , Osteogenesis , Rabbits , Real-Time Polymerase Chain Reaction , Tissue Scaffolds/chemistry , Tomography, X-Ray ComputedABSTRACT
Physiological osteochondral interface regeneration is a significant challenge. This study aims to investigate the effect of the coculture of chondrogenic rabbit bone marrow stromal cells (rBMSCs) with rabbit osteoblasts in a specially designed two-dimensional (2D)-three-dimensional (3D) co-interface culture to develop the intermediate osteochondral region in vitro. The 2D-3D coculture system was set up by first independently culturing chondrogenic rBMSCs on a scaffold and osteoblasts in cell culture plates, and subsequently placed in contact and cocultured. As control, samples not cocultured with osteoblasts were used. The regulatory effects exerted by osteoblasts on chondrogenic rBMSCs were quantified by real-time polymerase chain reaction. To study the effect of coculture on cells located in different parts of the scaffold, samples were separated into two parts and significantly different gene expression patterns were found between them. In comparison with the control group, a significant moderate downregulation of chondrogenic marker genes, such as Collagen II and Aggrecan was observed. However, the Sox-9 and Collagen I expression increased. More importantly, chondrogenic rBMSCs in the coculture system were shown to form the osteochondral interface layer by expressing calcified cartilage zone specific extracellular matrix marker Collagen X and the hypertrophic chondrocyte marker MMP-13, which were not observed in the control group. Specifically, only the chondrogenic rBMSC layer in contact with the osteoblasts expressed Collagen X and MMP-13, indicating the positive influence of the coculture upon interface formation. Biochemical analyses, histology results, and immunohistochemical staining further supported this observation. In conclusion, this study revealed that specific regulatory stimulations from osteoblasts in the 2D-3D interface coculture system could induce the formation of ostochondral interface for the purpose of osteochondral tissue engineering.