Cyclic mechanical loading enables solute transport and oxygen supply in bone healing: an in vitro investigation.

Bone healing is a complex process with an increased metabolic activity and consequently high demand for oxygen. In the hematoma phase, inflammatory cells and mesenchymal stromal cells (MSCs) are initially cut off from direct nutritional supply via blood vessels. Cyclic mechanical loading that occurs, for example, during walking is expected to have an impact on the biophysical environment of the cells but meaningful quantitative experimental data are still missing. In this study, the hypothesis that cyclic mechanical loading within a physiological range significantly contributes to oxygen transport into the fracture hematoma was investigated by an in vitro approach. MSCs were embedded in a fibrin matrix to mimic the hematoma phase during bone healing. Construct geometry, culture conditions, and parameters of mechanical loading in a bioreactor system were chosen to resemble the in vivo situation based on data from human studies and a well-characterized large animal model. Oxygen tension was measured before and after mechanical loading intervals by a chemical optical microsensor. The increase in oxygen tension at the center of the constructs was significant and depended on loading time with maximal values of 9.9%±5.1%, 14.8%±4.9%, and 25.3%±7.2% of normal atmospheric oxygen tension for 5, 15, and 30 min of cyclic loading respectively. Histological staining of hypoxic cells after 48 h of incubation confirmed sensor measurements by showing an increased number of normoxic cells with intermittent cyclic compression compared with unloaded controls. The present study demonstrates that moderate cyclic mechanical loading leads to an increased oxygen transport and thus to substantially enhanced supply conditions for cells entrapped in the hematoma. This link between mechanical conditions and nutrition supply in the early regenerative phases could be employed to improve the environmental conditions for cell metabolism and consequently prevent necrosis.

The impact of substrate stiffness and mechanical loading on fibroblast-induced scaffold remodeling.

Fibroblasts as many other cells are known to form, contract, and remodel the extracellular matrix (ECM). The presented study aims to gain an insight into how mechanical boundary conditions affect the production of ECM components, their remodeling, and the feedback of the altered mechanical cell environment on these processes. The influence of cyclic mechanical loading (f=1 Hz, 10% axial compression) and scaffold stiffness (E=1.2 and 8.5 kPa) on the mechanical properties of fibroblast-seeded scaffold constructs were investigated in an in vitro approach over 14 days of culture. To do so, a newly developed bioreactor system was employed. While mechanical loading resulted in a clear upregulation of procollagen-I and fibronectin production, scaffold stiffness showed to primarily influence matrix metalloproteinase-1 (MMP-1) secretion and cell-induced scaffold contraction. Higher stiffness of the collagen scaffolds resulted in an up to twofold higher production of collagen-degrading MMP-1. The changes of mechanical parameters like Young's modulus, maximum compression force, and elastic portion of compression force over time suggest that from initially distinct mechanical starting conditions (scaffold stiffness), the construct's mechanical properties converge over time. As a consequence of mechanical loading a shift toward higher construct stiffness was observed. The results suggest that scaffold stiffness has only a temporary effect on cell behavior, while the impact of mechanical loading is preserved over time. Thus, it is concluded that the mechanical environment of the cell after remodeling is depending on mechanical loading rather than on initial scaffold stiffness.

Characterization of a rat osteotomy model with impaired healing.

BACKGROUND: Delayed union or nonunion are frequent and feared complications in fracture treatment. Animal models of impaired bone healing are rare. Moreover, specific descriptions are limited although understanding of the biological course of pathogenesis of fracture nonunion is essential for therapeutic approaches. METHODS: A rat tibial osteotomy model with subsequent intramedullary stabilization was performed. The healing progress of the osteotomy model was compared to a previously described closed fracture model. Histological analyses, biomechanical testing and radiological screening were undertaken during the observation period of 84 days (d) to verify the status of the healing process. In this context, particular attention was paid to a comparison of bone slices by histological and immunohistological (IHC) methods at early points in time, i.e. at 5 and 10 d post bone defect. RESULTS: In contrast to the closed fracture technique osteotomy led to delayed union or nonunion until 84 d post intervention. The dimensions of whole reactive callus and the amounts of vessels in defined regions of the callus differed significantly between osteotomized and fractured animals at 10 d post surgery. A lower fraction of newly formed bone and cartilaginous tissue was obvious during this period in osteotomized animals and more inflammatory cells were observed in the callus. Newly formed bone tissue accumulated slowly on the anterior tibial side with both techniques. New formation of reparative cartilage was obviously inhibited on the anterior side, the surgical approach side, in osteotomized animals only. CONCLUSION: Tibial osteotomy with intramedullary stabilisation in rats leads to pronounced delayed union and nonunion until 84 d post intervention. The early onset of this delay can already be detected histologically within 10 d post surgery. Moreover, the osteotomy technique is associated with cellular and vascular signs of persistent inflammation within the first 10 d after bone defect and may be a contributory factor to impaired healing. The model would be excellent to test agents to promote fracture healing.