Simulation of cell differentiation in fracture healing: mechanically loaded composite scaffolds in a novel bioreactor system.

Cell differentiation during bone healing following a fracture is influenced by various biological and mechanical factors. We introduce a method for the examination of cell and tissue differentiation simulating a fracture gap in vitro. A closed bioreactor system allows the imitation of the biological, mechanical, and biochemical conditions in vitro. The initial hematoma formed in a fracture is simulated with a mixed construct composed of lyophilized cancellous bone and a fibrin matrix in a sandwich configuration. The construct may be loaded with osteoprogenitor cells. Exemplarily, constructs were loaded with rabbit periosteal cells and cultivated under mechanical loading with 7 kPa at 0.05 Hz for up to two weeks. During the observation period, cell morphology and correlating protein synthesis changed under mechanical stimulation. Cell differentiation differed between the various regions of the constructs. The periosteal cells were arranged perpendicularly to the mechanical loading and differentiated to osteoblastic forms with rising collagen type I synthesis, constant alkaline phosphatase activity, and initiation of the calcification of the extracellular matrix. The observed pattern of cell and tissue differentiation was similar to the one seen in the early phase of bone healing. In conclusion, the presented method allows simulation of cell and tissue differentiation during the early phase of fracture healing. It could serve as an in vitro model for the examination of mechanical and pharmacological influences during the early phase of bone healing on a cellular level

Mesenchymal stem cells regulate angiogenesis according to their mechanical environment.

In fracture and bone defect healing, MSCs largely drive tissue regeneration. MSCs have been shown to promote angiogenesis both in vivo and in vitro. Angiogenesis is a prerequisite to large tissue reconstitution. The present study investigated how mechanical loading of MSCs influences their proangiogenic capacity. The results show a significant enhancement of angiogenesis by conditioned media from mechanically stimulated compared with unstimulated MSCs in two-dimensional tube formation and three-dimensional spheroid sprouting assays. In particular, proliferation but not migration or adhesion of endothelial cells was elevated. Promotion of angiogenesis was dependent upon fibroblast growth factor receptor 1 (FGFR1) signaling. Moreover, stimulation of tube formation was inhibited by vascular endothelial growth factor receptor (VEGFR) tyrosine kinase blocking. Screening for the expression levels of different soluble regulators of angiogenesis revealed an enrichment of matrix metalloprotease 2, transforming growth factor beta1, and basic fibroblast growth factor but not of vascular endothelial growth factor in response to mechanical stimulation. In conclusion, mechanical loading of MSCs seems to result in a paracrine stimulation of angiogenesis, most likely by the regulation of a network of several angiogenic molecules. The underlying mechanism appears to be dependent on the FGFR and VEGFR signaling cascades and might be mediated by an additional cross-talk with other pathways