(55e) Effect of TNF-Alpha On in Vitro Osteogenic Differentiation of Mesenchymal Stem Cells

Craniofacial bone has the remarkable capacity to heal without scarring, but this regeneration process fails in bone defects above a critical size. The limitations of current surgical reconstruction techniques have led to increased interest in craniofacial tissue engineering. Informed by cell biology research, efforts to date have focused on inducing bone regeneration via delivery of various bioactive molecules, particularly osteogenic factors. However, recent insight into the critical role of pro-inflammatory cytokines, particularly tumor necrosis factor alpha (TNF-alpha), in bone healing has heralded a new direction in the rational design of bone tissue engineering constructs. The goal of this study was to characterize the impact of varying TNF-alpha concentrations (0, 0.1, 5, and 50 ng/mL) and exposure durations (from 0-16 d) on osteogenic differentiation of rat mesenchymal stem cells (MSCs) grown in a 3D culture system. MSCs were cultured in 3D biodegradable electrospun poly(epsilon-caprolactone) scaffolds with pregenerated bone-like extracellular matrix (ECM), a novel culture system developed in our laboratory. Pregenerated bone-like ECM has the advantage of inducing MSC osteogenic differentiation without the need for the osteogenic cell culture supplement dexamethasone, an anti-inflammatory agent that might mask the effects of TNF-alpha. Differentiating MSCs were exposed to various doses of TNF-alpha, for varying lengths of time. Biochemical assays were used on days 0, 4, 8, and 16 to determine the effect of TNF-alpha on markers of MSC osteogenic differentiation, including extent of new mineralized ECM production. To our knowledge, this is the first study to explore the biological effects of TNF-alpha, an inflammatory signal, on osteogenesis in a 3D culture model. Through rational control of inflammation, craniofacial bone regeneration can be augmented and potentially accelerated. The information gained from these studies will enable the design of tissue engineering constructs with a better combination of renewal signals relevant for regeneration of large critical size cranial defects. Moreover, this research will provide a powerful strategy to modulate biological responses and induce healing of engineered tissue.