Bone healing outcome is highly dependent on the initial mechanical fracture environment [1]. In vivo, direct bone healing requires absolute stability and an interfragmentary strain (IFS) below 2% [2]. In the majority of cases, however, endochondral ossification is engaged where frequency and amplitude of IFS are key factors. Still, at the cellular level, the influence of those parameters remains unknown. Understanding the regulation of naïve hMSC differentiation is essential for developing effective bone healing strategies. Human bone-marrow-derived MSC (KEK-ZH-NR: 2010–0444/0) were embedded in 8% gelatin methacryol. Samples (5mm Ø x 4mm) were subjected to 0, 10 and 30% compressive strain (5sec compression, 2hrs pause sequence for 14 days) using a multi-well uniaxial bioreactor (RISystem) and in presence of chondro-permissive medium (CP, DMEM HG, 1% NEAA, 10 µM ITS, 50 µg/mL ascorbic acid, and 100 mM Dex). Cell differentiation was assessed by qRT-PCR and histo-/immunohistology staining. Experiments were repeated 5 times with cells from 5 donors in duplicate. ANOVA with Tukey post-hoc correction or Kurskal-Wallis test with Dunn's correction was used. Data showed a strong upregulation of hypertrophic related genes COMP, MMP13 and Type 10 collagen upon stimulation when compared to chondrogenic SOX9, ACAN, Type 2 collagen or to osteoblastic related genes Type 1 Collagen, Runx2. When compared to chondrogenic control medium, cells in CP with or without stimulation showed low proteoglycan synthesis as shown by Safranine-O-green staining. In addition, the cells were significantly larger in 10% and 30% strain compared to control medium with 0% strain. Type 1 and 10 collagens immunostaining showed stronger Coll 10 expression in the samples subjected to strain compared to control. Uniaxial deformation seems to mainly promote hypertrophic-like chondrocyte differentiation of MSC. Osteogenic or potentially late hypertrophic related genes are also induced by strain.
Despite its clinical significance, metaphyseal fracture healing has received little attention in research and experimental models have been limited. In particular it is not known to what extent the mechanical environment plays a role in metaphyseal fracture healing. Recently, a new murine internal fixation plate has been developed to stabilise fractures in the distal femur under highly standardised conditions. Goal of the current study was to modify this design, in order to be able to evaluate the influence of the fixator bending stiffness on metaphyseal fracture healing in mice. Adapting the existing single body design, resulting in low flexibility fixation, two new plates were developed with a decreased bending stiffness of approximately 65% and 45% of the original implant (100%). Pilot experiments were performed on 54 animals, whereas the mice were sacrificed and fracture healing assessed radiologically and biomechanically after 14 and 28 days. MicroCT evaluation confirmed that the osteotomy was created in the trabecular, metaphyseal bone of the distal mouse femora. All bones showed progressive fracture healing over time, with decreased implant stiffness leading to increased periosteal callus formation. These implants represent an important new research tool to study molecular and genetic aspects of metaphyseal fracture healing in mice under standardized mechanical conditions, in order to improve clinical treatment in challenging situations, such as in osteoporotic bone.
During the last decades numerous studies have reported the critical impact of physical activity on bone repair. While most studies have evaluated the tissue response to the local mechanical environment within the fracture gap, there is a lack of information on the systemic role of physical activity during fracture healing. Therefore, the aim of this study was to standardize the mechanical environment in the fracture gap by developing a rotationally and axially stable murine fracture model, and thereby to analyze the systemic influence of physical activity on early bone repair. After stable fixation of a closed femoral fracture, mice (n=18) were housed in cages supplied with running wheels (running distance >
500m/d). At 2 weeks animals were sacrificed and bones were prepared for histomorphometric (n=7), biomechanical (n=7), and protein biochemical analyses (n=4). Additional mice (n=22), which were housed in standard cages, served as controls. Histomorphometric evaluation showed no influence of increased physical activity on bone repair in terms of callus size and tissue composition. Accordingly, also biomechanical testing of the callus revealed no differences between both groups in rotational stiffness, peak rotation angle, and load at failure. Western blot analyses demonstrated no alterations in callus expression of proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF) after daily running when compared to controls. We conclude that increased physical activity under standardized mechanical conditions in the fracture gap does not affect early bone repair in mice.
