(G-275) The Impact of Rehabilitation Parameters on Bone Healing Depends on Injury Size

Introduction:: Complex bone injuries exhibit high complication rates and poor functional recovery, partially due to insufficient understanding of how rehabilitative loading impacts bone healing. The emerging field of regenerative rehabilitation aims to uncover the relationship between regenerative medicine and rehabilitation strategies by exploring how mechanical stimuli impact the ability for tissues to regenerate. Within this field, our lab investigates rehabilitation regimens that may aid in functional regeneration of bone healing in our rodent segmental defect model. Despite the general understanding that loading increases bone formation and promotes healing (Klosterhoff et al. 2020; Boerckel et al. 2011; Goodship and Kenwright 1985), literature has also found that too much loading has been detrimental to bone healing (Boerckel et al. 2011). This highlights the lack of knowledge regarding the optimal amount of loading and its translation into rehabilitation decisions. With this in mind, we aimed to study the impact of rehabilitative running on the healing of bone injuries. Our study gave rats voluntary access to running for either a 24/7 or restricted 2 hrs/day duration to investigate how exercise duration impacts bone healing after surgically induced femoral injuries. We hypothesized that restricted rehabilitation (2 hrs/day of wheel access) would increase bone formation in an injury size dependent manner.

Materials and Methods:: To investigate the effects of rehabilitation parameters on bone healing of different injury sizes, we assessed longitudinal activity data and bone healing after surgery to create bilateral segmental 2 and 3mm femoral defects that were stabilized with compliant fixation plates. A total of 20 rats were included across sedentary (n = 7), 2 hrs/day of wheel access (n = 7), and 24/7 of wheel access (n = 6). In hopes of creating a low versus high activity group, we gave one group 24/7 access to wheels (Exotic Nutrition Store) and another group only 2 hrs/day of wheel access. Rats in the sedentary group were housed in standard cages with no wheel, while the 2hrs/day of wheel access subjects were housed in standard sedentary cages and manually transferred to wheel cages from 8-10pm daily. Subjects in the 24/7 wheel access group were housed in a wheel cage, allowing for unlimited access to a wheel. In-vivo radiography and microCT scans were performed at two, four, and eight-week time points to monitor longitudinal bone healing. Continuous activity data were also collected using revolution counters. Lafayette Scurry software and Python code were used to calculate activity parameters (distance, duration, number of running bouts, rest time, etc). Statistical analysis involved Mann Whitney U-test to analyze the significant impact of rehabilitation conditions and time on daily running bouts and maximum rest time. Additionally, two-way ANOVA with Tukey's multiple comparisons test was used to analyze the significant impact of rehabilitation conditions and time on bone formation.

Results, Conclusions, and Discussions:: Analysis of longitudinal activity data revealed that surprisingly both rehabilitation groups ran the same daily distance (m/day) and running duration (hrs/day) independent of hours of wheel access (Figure 1A). We believe this is due to several factors, including (1) the 2 hrs/day of wheel access group could’ve been well rested, allowing them to run “harder” during their restricted running window or (2) this group was behaviorally more excited to run since they learned they had a restricted window of access to the wheels. The group with 24/7 wheel access exhibited significantly greater running bouts per day (Mann Whitney t-test, *** p = 0.0006) and significantly less rest time (Mann Whitney t-test, *** p = 0.0006) compared to the group with only 2 hours of daily wheel access (Figure 1A). We also found that the 24/7 treadwheel access group ran more bouts/day, at a slower velocity than the 2 hrs/day group and decided to rest for a maximum of 7 hrs/day (Figure 1A). MicroCT 3D reconstructions of 3mm explanted femurs revealed that no subjects in the 24/7 wheel access group bridged, while the 2 hrs/day of wheel access group exhibited more uniform bridging than femurs from sedentary subjects (Figure 1B). Further, microCT quantification revealed that 24/7 treadwheel access, which resulted in subjects training more often and resting for fewer hours each day, exhibited significantly less bone formation than the group with 2 hrs/day of wheel access (p = 0.0063) and sedentary counterparts (p = 0.0192, Figure 1B). In contrast, for the 2mm injuries, we found that there was no difference in the bone volume or bridging integrity (Figure 1C). Overall, the bridging rates of the 24/7 wheel access group changed with injury severity; 3mm bone injuries had a 0% bridging rate, while 2mm bone injuries had an 80% bridging rate (Figure 2). Ultimately, our study suggests that optimized rehabilitation protocols depend on patient-specific factors such as injury severity. For more severe injuries, limiting exercise and ensuring adequate rest is critical to avoid negative effects on bone healing. In contrast, for less severe injuries, rehabilitation exercise improved bridging rates.

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