Early Rehabilitation Promotes Functional Recovery In Segmental Bone Defects

Bone defect repair is a significant clinical burden, often resulting in prolonged functional deficits. The standard of care for these injuries is stabilization with a load-shielding fixation plate coupled with an autologous bone graft and eventual rehabilitation. For more severe defects and open fractures, this approach can require multiple revision surgeries and results in chronic pain and disability in patients. Although mechanical loading is integral to bone maintenance and repair, the load-shielding fixation plate prevents mechanical stimulation across the bone defect. In addition, current clinical rehabilitation regimens typically involve long periods of non-loading and rely on subjective patient feedback throughout the healing process. Several pre-clinical studies have demonstrated that modulating the stiffness of the fixation plate to allow for increased mechanical loading across the defect can improve healing outcomes. However, there is not yet a consensus on the impacts of early rehabilitation on bone healing, functional recovery, and mechanoresponsive signaling mechanisms. The objective of this research was to investigate how rehabilitation impacts bone regeneration, hindlimb functional recovery, and mechanotransductive gene expression in a segmental bone defect. Our hypothesis was that initiation of rehabilitation at seven days after injury would improve functional bone recovery and promote mechanosensitive gene expression.

All procedures were conducted in accordance with approved IACUC protocol. Mice were given voluntary access to running wheels, acclimated for two weeks, and then the wheels were removed on the day of surgery. A 1mm femoral defect was surgically induced in the left hindlimb of female, 12-week-old C57BL/6 mice (Jackson Labs). Defects were stabilized by an external fixation plate with a stiffness of 18.1N/mm (MouseExFix, RISystem), and mice were divided into sedentary and rehabilitation groups. Wheels were reintroduced for the group undergoing rehabilitation one week after surgery and daily distance was monitored. Bridging was monitored longitudinally via radiographs and evaluated by blinded scorers at weeks 2, 4, 6, and 8. Hindlimb sensitivity was evaluated using functional testing, including von Frey testing, at baseline and weeks 1, 2, 3, 4, 6, and 8. At the end of the study, muscle strength was assessed using maximum isometric force testing, and bone volume, mineralization, and callus size were evaluated via micro-computed tomography (µCT). To assess functional bone strength and tissue composition, torsional testing and histological analyses will be performed. Forthcoming data to evaluate gene expression will be assessed via takedowns prior to week 4. Data are presented as the mean ± standard error of the mean. Longitudinal results were analyzed with a two-way ANOVA and Tukey post-hoc analysis for pairwise comparisons (95%), while individual timepoints were analyzed with an unpaired t-test using GraphPad Prism 9.

Daily running increased during the two weeks of pretraining until reaching an average of 18 km/day (Fig. 1A). Once running wheels were reintroduced after surgery, daily activity was reduced to an average of 6.5 km/day. Running distance increased through week 6 when it reached a peak of 16 km/day, before dropping to 12 km/day for the last two weeks. Radiographs and µCT 3D reconstructions at week 8 showed 4/6 bridged in the rehabilitation group compared to 3/6 bridged in the sedentary group, two of which were poorly bridged (Fig. 1B). Hindlimb sensitivity increased after injury, then returned to baseline levels by week 3 (Fig. 1C). At two weeks post injury, subjects undergoing rehabilitation had reduced mechanical allodynia compared to sedentary subjects (36.90% withdrawal ± 3.41 vs. 46.43% withdrawal ± 2.44, p=0.046) (Fig. 1D). Bone volume at 8 weeks post injury was not statistically different between groups (sedentary: 0.2668 mm³ ± 0.090 vs. 0.3907 mm³ ± 0.113). Maximum isometric torque analysis showed that both groups had comparable muscle strength that was not different to that of the contralateral limb.

Early changes in running activity coincide with injury and initial healing. The decrease between weeks 6 and 7 coincide with a change in housing, indicating increased stress in mice when housing locations are changed. Analysis of von Frey showed increased sensitivity following injury, and recovery by week 3 after injury for both groups. Analysis at week 2 indicated that the rehabilitation group reached baseline levels of sensitivity sooner than the sedentary group. In addition, while both groups had comparable regenerated bone volume and bridging rates, qualitative evaluation of radiographs and µCT reconstructions show a more structurally sound bridging in the rehabilitation subjects (Fig. 1B). Mechanical testing and histological analyses will provide further insight into the impacts of rehabilitation on the quality of the regenerated bone. These results indicate that early initiation of rehabilitation may be advantageous to restoring functionality when compared to conservative rehabilitation levels. Forthcoming data into gene expression levels will inform the changes at the cellular and molecular level that drive these changes in function.