Cellular activation status in femoral shaft fracture hematoma following different reaming techniques – A large animal model

Abstract The local inflammatory impact of different reaming protocols in intramedullary nailing has been sparsely investigated. We examined the effect of different reaming protocols on fracture hematoma (FH) immunological characteristics in pigs. To do so, a standardized midshaft femur fracture was induced in adult male pigs. Fractures were treated with conventional reamed femoral nailing (group RFN, n = 6); unreamed femoral nailing (group UFN, n = 6); reaming with a Reamer Irrigator Aspirator device (group RIA, n = 12). Animals were observed for 6 h and FH was collected. FH‐cell apoptosis and neutrophil receptor expression (Mac‐1/CD11b and FcγRIII/CD16) were studied by flow cytometry and local temperature changes were analyzed. The study demonstrates that apoptosis‐rates of FH‐immune cells were significantly lower in group RIA (3.50 ± 0.53%) when compared with non‐RIA groups: (group UFN 12.50 ± 5.22%, p = 0.028 UFN vs. RIA), (group RFN 13.30 ± 3.18%, p < 0.001, RFN vs. RIA). Further, RIA‐FH showed lower neutrophil CD11b/CD16 expression when compared with RFN (mean difference of 43.0% median fluorescence intensity (MFI), p = 0.02; and mean difference of 35.3% MFI, p = 0.04, respectively). Finally, RIA induced a transient local hypothermia and hypothermia negatively correlated with both FH‐immune cell apoptosis and neutrophil activation. In conclusion, immunologic changes observed in FH appear to be modified by certain reaming techniques. Irrigation during reaming was associated with transient local hypothermia, decreased apoptosis, and reduced neutrophil activation. Further study is warranted to examine whether the rinsing effect of RIA, specific tissue removal by reaming, or thermal effects predominantly determine local inflammatory changes during reaming.


| METHODS
The current study was approved by the local Official Veterinary Office (Kanton Zürich, Gesundheitsdirektion Veterinäramt under Project number: ZH138/17). All animal experiments were designed and carried out in accordance with the "Guide for Care and Use of Laboratory Animals." 19 Data processing and documentation have been performed in line with the ARRIVE guidelines for reporting animal research. 20

| Experimental model and study groups
A standardized animal protocol was applied, as previously described. 10,12,17 Briefly, experiments were performed using 24 male Swiss Large White pigs (4 months old animals weighing 50 ± 5 kg). Before the start of the experiment, animals received pre-medication by in- Then, all animals underwent standardized unilateral femoral fracture as previously described. 10,12,17 Briefly, a bolt gun machine (Blitz-Kerner, turbocut JOBB GmbH) and a custom-made metal plate were applied to produce a standardized midshaft transverse femur fracture. Fracture location and morphology were confirmed by fluoroscopy.
In the first 90 min after trauma, the preclinical phase was mimicked with altered ventilator settings, no temperature correction, reduced fluid infusion of 10 ml/h, and FiO 2 reduction to ambient air oxygen level of 0.21.

| Surgical intervention
The standardized nailing system utilized a tailored 120 mm nail (cannulated DFN Ø 10.0 mm, DePuy Synthes). In animals randomized for RFN, standardized stepwise intramedullary reaming preceded nailing. The SynReam intramedullary reaming system (DePuy Synthes) was utilized and reamer heads with a diameter up to 12 mm were used. In the RIA group, a Synthes Reamer/Irrigator/Aspirator System (DePuy Synthes) was used according to protocol. Again, reamer heads of 12 mm were utilized.
Reamer heads were replaced after every five experiments in all study groups. Sequential reaming at intervals of 0.5 mm was performed. Final reaming with the 12 mm drill head was repeated twice. To optimize standardization of our study conditions, all fractures treated by a treatment modality including intramedullary nailing, have been reamed 10 times in total. The experimental design is presented as a flowchart in

| Systemic body temperature regulation
Core temperature (T core ) was monitored by using an esophageal temperature probe connected to a Datascope Passport2 Patient Monitoring System (Pacific Medical). Core temperature was cor- were compared between groups.

| Termination and tissue collection
Animals were terminated after an observation period of 6 h with a Na-pentobarbital (Esconarkon ad us. Vet., Streuli Pharma AG) infusion. Before termination, samples of FH were collected. This receptor binds to immunoglobulins (IgG) either in aggregates or attached to pathogens. Binding of IgG to Fcγ receptors promotes the oxidative burst and induces phagocytosis. 24 Short term in vitro activation of neutrophils by lipopolysaccharide is associated with an initial increase in neutrophil CD16-expression. 25 Neutrophil FcγRIIIreceptor expression increases during cell maturation, with young neutrophils typically having relatively low CD16-cell surface expression compared to older counterparts. 26,27 CD45 is a pan-leukocyte marker and is utilized to identify immune cells in blood and the tissue compartment. 28 FlowJo (Becton & Dickinson) was utilized for the evaluation of flow cytometry data and the gating strategy utilized is summarized in Supporting Information 1. Leukocyte subtype identification is based on forward-sideward scatter gating and CD45-expression levels. This protocol has previously been validated in pilot experiments using both porcine blood and hematoma samples. Differential cell counts were obtained with cytospin-prepared slides stained with May-Grünwald-Giemsa.

| Sample size calculation
Sample size calculations were based on tissue neutrophil presence after intervention. In a previous study on pulmonary neutrophil migration after polytrauma performed by this study group, 12 animals were exposed to polytrauma while 6 animals were included in a sham control group. Neutrophil presence in lung tissue in the intervention group (n = 12) was determined as 2.38 (SD: 0.76) cells per high power field, whereas 0.98 (SD: 0.2) cells/high power field were counted in the sham group (n = 6). This results in power of 98% if the sample size in two groups is 4 at a 5% significance level. 29 However, the number of animals and the allocation ratio of 2:1:1 in the current study were based primarily on logistic and ethical considerations instead of a formal a-priori sample size calculation.
Sample sizes of 12 and 6 in the groups were used to avoid an underpowered study protocol.

| Statistical analysis
All statistical analyses were performed using GraphPad Prism Version 7.
Unpaired t-tests (normally distributed datasets) or Mann-Whitney U-tests (nonparametrical analysis) were performed. Correlation analysis and plotting were performed with Excel (Microsoft) and a web-based Pearson Correlation Coefficient Calculator, retrieved from Social Science Statistics at www.socscistatistics.com. A p value < 0.05 was considered statistically significant. All data are presented as standard error of the mean (SEM).

| RESULTS
All 24 animals survived the observation period. No intra-or postoperative complications were observed.   Excessive systemic immune activation precedes inadequate bone healing by altering the immunological composition of early FH. [31][32][33][34] Specifically, studies suggest that excessive influx of activated neutrophils into FH is associated with inadequate fracture healing. 11 Despite varying reaming strategies, PMN levels in FH were consistent across all groups in the current study. Interestingly, however, while no quantitative differences in immune cell composition were found, prominent variations in FH neutrophil activation status were encountered between groups.  Further, conventional RFN was associated with enhanced FHneutrophil CD16-receptor expression, which is considered an essential receptor in the phagocytic process. 44 Decreased PMN-CD16-expression after RIA may be due to the influx of immature band neutrophils from the bone marrow. 45 These study results must, however, be qualified considering several possible limitations. First, this experiment focused on the early cellular immune reaction at the fracture site, which is dominated by neutrophils. Long-term effects of different reaming protocols were therefore not investigated. Furthermore, to avoid artificial manipulation at the fracture site, local temperature probes were not inserted directly into the fracture but were fluoroscopically guided to a standardized position near the fracture site.

| FH-immune cell subpopulation composition
As a result, FH temperature measurements in this study were indirect, but consistent approximations across all study groups.