Developing a viable and functional bone scaffold in vitro that is capable of surviving and bearing mechanical load in vivo requires an understanding of the cell biology of osteoprogenitor cells, particularly how they are influenced by mechanical stimulation during cell differentiation and maturation. In this study, mechanical load was applied using a modified FlexCell plate to impart confined compression to collagen-I scaffolds seeded with undifferentiated murine embryonic stem cells. The activity, presence, and expression of osteoblast-cadherin (OB-Cad) and connexin-43, as well as various pluripotent and osteogenic markers were examined at 5-30 days of differentiation as cells were stimulated to differentiate to osteoblasts with and without applied mechanical load. Fluorescence recovery after photobleaching, immunofluorescence, viability, von Kossa, and real-time polymerase chain reaction assessments revealed that mechanical prestimulation of this cell-seeded scaffold altered the expression of OB-Cad and connexin-43 and resulted in significant differences in the structure and organization of mineralization present in the collagen matrix. Specifically, cells in gels that were loaded for 40 h after 5 days of differentiation and then left to fully differentiate for 30 days produced a highly structured honeycomb-shaped mineralization in the matrix; an outcome that was previously shown to be indicative of late osteoblast/early osteocyte activity. This study highlights the potential of mechanical load to accelerate differentiation and enhance osteoblast communication and function during the differentiation process, and highlights a time point of cell differentiation within this scaffold to apply load in order to most effectively transduce a mechanical signal.
Fracture nonunions represent one of many large bone defects where current treatment strategies fall short in restoring both form and function of the injured tissue. In this case, the use of a tissue-engineered scaffold for promoting bone healing offers an accessible and easy-to-manipulate environment for studying bone formation processes in vitro. We have previously shown that mechanical prestimulation using confined compression of differentiating osteoblasts results in an increase in mineralization formed in a 3D collagen-I scaffold. This study builds on this knowledge by evaluating the short and long-term effects of blocking gap junction-mediated intercellular communication among osteogenic cells on their effectiveness to mineralize collagen-I scaffolds in vitro, and in the presence and absence of mechanical stimulation. In this study, confined compression was applied in conjunction with octanol (a general communication blocker) or 18-α-glycerrhetinic acid (AGA, a specific gap junction blocker) using a modified FlexCell plate to collagen-I scaffolds seeded with murine embryonic stem cells stimulated toward osteoblast differentiation using beta-glycerol phosphate. The activity, presence, and expression of osteoblast cadherin, connexin-43, as well as various pluripotent and osteogenic markers were examined at 5-30 days of differentiation. Fluorescence recovery after photobleaching, immunofluorescence, viability, histology assessments, and reverse-transcriptase polymerase chain reaction assessments revealed that inhibiting communication in this scaffold altered the lineage and function of differentiating osteoblasts. In particular, treatment with communication inhibitors caused reduced mineralization in the matrix, and dissociation between connexin-43 and integrin α5β1. This dissociation was not restored even after long-term recovery. Thus, in order for this scaffold to be considered as an alternative strategy for the repair of large bone defects, cell-cell contacts and cell-matrix interactions must remain intact for osteoblast differentiation and function to be preserved. This study shows that within this 3D scaffold, gap junctions are essential in osteoblast response to mechanical loading, and are essential structures in producing a significant amount and organization of mineralization in the matrix.
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