M2 macrophages are closely associated with accelerated clavicle fracture healing in patients with traumatic brain injury: a retrospective cohort study

This study was conducted under the guide of the Declaration of Helsinki and approved by the Ethical Review Committee of the Liuzhou General Hospital. Written informed consent was obtained from all subjects prior to their participation in the study. Forty-seven patients with clavicle fracture were admitted to the Department of Orthopedics, Liuzhou General Hospital, between January 2009 and July 2017. The fractures in all patients were treated operatively. Twenty-five patients with clavicle fracture alone (CF group) underwent open reduction and internal fixation of the clavicle within 1 or 2 weeks after injury. Twenty-two patients with clavicle fracture and concomitant TBI (CFT group) consisted of 20 cases caused by traffic accidents and 2 cases caused by fall injury. Among these 22 patients, 12 patients with TBI were treated with open reduction and internal fixation of the clavicle within 1 week post-injury and 10 patients underwent surgery between 7 and 14 days after brain injury. After admission, the patients of two groups were both examined by conventional X-rays for the clavicle fracture and evaluated with the Glasgow Coma Scale (GCS) system. Callus volume measurement of bone fracture before operation, length, width, and height, was obtained from computed tomography (CT) scan images before surgery. We use the following formula to calculate the callus volume: volume = length × width × height × π/6, and the width and height of callus tissue in the formula were subtracted the width and height of clavicle bone respectively. The CFT group was diagnosed with computerized tomography of the head and the GCS score is less than 8 points, while the CF group without TBI, the GCS score is more than 13 points. Participants with any forms of prior nervous system or bone-related diseases, multiple fractures, autoimmune disease, malignant disease, diabetes, rheumatoid arthritis, or other chronic inflammation diseases as well as history of long time steroid, non-steroidal anti-inflammatory drugs, and immunosuppressant or bisphosphonate therapy or survived less than 1 year after surgery were excluded.

All patients were followed up for at least 24 weeks after the operation. The follow-up examination was based on clinical and radiological examination at 1, 2, 4, 8, 12, 16, 20, and 24 weeks after trauma. The fracture was recorded as clinical heal based on the following clinical indications: (1) the patient had no pain at the surgical site and no tenderness; (2) the affected side could lift a 1-kg weight for 1 min; and (3) the X-ray photographs showed that the fracture line was blurred and there was a continuous epiphyseal fracture line. Two radiologists, who were blinded to the presence or absence of TBI, independently evaluated the bone healing.

During the operation, the callus tissue between fractures was examined by immunohistochemistry with anti-CD68 (MXB Biotechnology, cat#: Kit-0026), anti-COX-2 (MXB Biotechnology, cat#: RMA-0549), and anti-CD206 (Santa Cruz Biotechnology, cat#: sc-376108). After antigen repair, H₂O₂ was added to the section and incubated for 10 min at room temperature to block the endogenous peroxidase; the PBS buffer was used to wash for 3 min × 3 times. One hundred microliters of the blocking normal calf serum working solution was added and incubated for 15 min at room temperature. After removing the serum, add 100 μl of primary antibody and store overnight at 4 °C. After warming at room temperature for 15 min on the second day, the PBS buffer was used to wash for 3 min × 3 times. One hundred microliters or an appropriate amount of biotin-labeled goat anti-mouse/rabbit IgG polymer was added and incubated at room temperature for 13 min; phosphate-buffered saline (PBS) buffer was used to wash for 3 min × 3 times. Add 100 μl or an appropriate amount of horseradish peroxidase-labeled streptolysin working solution and incubate at room temperature for 10 min; rinse with PBS buffer for 3 min × 3 times. An appropriate amount of freshly prepared DAB was added and incubated for 5–8 min. Counterstaining was performed using tap water, and hematoxylin stain was incubated for 20 s; differentiation and flushing returned to blue.

Immunohistochemistry was performed on each slice, and five fields of view were selected from the top left, top right, bottom right, bottom left, and the middle and were imaged with the Minmei microscope digital imaging system (V9.5.2). The images were analyzed with Image-pro plus 6.0 (Media Cybernetics, Rockville, MD, USA). The immunohistochemistry images were opened in Image-pro plus software. Using count and measure functions, positive cells were manually selected in several images in order to train the software. After that, all positive cells were automatically counted and processed by the software while the sensitivity was set to level 4.

Statistical analysis was performed with the Statistical Package for Social Sciences software (SPSS Inc.; Chicago, IL, USA), version 16.0 for Windows. All data were presented as mean, standard deviation of the mean (SD), or median (interquartile range). Kolmogorov-Smirnov test was performed to analyze the data normality, and Student’s t test, Mann-Whitney U test, or Chi-square test were used to assess the significance in clinical characteristics between patients with fracture only and concomitant TBI. Pearson correlation was used to assess significance among healing time and clinical variables. p < 0.05 was considered to be statistically significant for differences and correlations.

Total of 25 patients with fracture alone (CF group) and 22 patients with fracture and concomitant TBI (CFT group) were included in this study. The demographics and clinical profile of patients in CF and CFT groups are summarized in Table 1. There are no significant differences in baseline clinical parameters, including age, gender, fracture place, and time from injury to surgery between the two groups (for all, p > 0.05). The CFT group has significantly lower GCS scores (11.5 vs 15,) and blood monocyte (0.058 vs 0.922) (Table 1), larger fracture callus volume (21.9 vs 8.5 cm³) (Fig. 1a), and shorter healing time (82.2 vs 127.0 days) (Fig. 1b) compared with the CF group (for all, p < 0.001). Callus volume is a very important indicator of bone fracture healing. If the other indicators are similar, the larger the size of the callus at the injury site, the faster the fracture healing. The preoperative photographs of X-ray showed the healing process of clavicle fractures in both groups, and the photographs of X-ray at 12 weeks after surgery showed that the fracture recovery was much faster in the CFT group than in the CF group (Fig. 2).

To characterize the relationship between macrophages and fracture healing, we measured the expression of COX-2 (M1-type macrophage marker), CD206 (M2-type macrophage marker), and CD68 (all macrophages marker) in the callus tissues after bone fracture for 1 week and 2 weeks with immunohistochemistry. Each section was randomly chosen five fields to be photographed, and the images were analyzed with Image-pro plus software. The percentages of M1-type macrophages decreased after bone fracture for 1 week and 2 weeks in both the CF group and CFT group (p < 0.001) (Figs. 3a and 4a), while the percentages of M2-type macrophages increased over time (Figs. 3b and 4b).

The percentages of M1-type macrophage, COX-2 positive macrophages, did not show significant difference between the CF and CFT groups of patients who underwent surgery within 1 week or between 8 and 14 days after injury (p > 0.05) (Figs. 3a and 4a), but the percentages of M1-type macrophage in both CF and CFT groups of patients who underwent surgery between 8 and 14 days after injury were significantly decreased, compared to that of patients who underwent surgery within 1 week after injury. The percentages of M2-type macrophages are significantly increased in both the CF group and CFT group of patients with time before surgery. Meanwhile, the increase of percentages of M2-type macrophages was significantly higher in the CFT group of patients than that in the CF group of patients who took surgery within 1 week (22.27% vs 11.21%, p < 0.001) and between 8 and 14 days (55.75% vs 26.25%, p < 0.001) after injury, respectively (Figs. 3b and 4b). In addition, the CD68 positive macrophages are not significantly different from these two groups over time (Fig. 4c). There is a significant negative correlation between fracture healing time and the percentage of M2-type macrophages in callus tissues (r = − 0.575, p < 0.01) (Fig. 5 and Table 2). The fracture healing time is also shown to be positively correlated with GCS scores and monocyte percentage in blood and negatively correlated with the callus volume (Table 2, p < 0.01). but not the percentage of M1-type macrophages, age, gender, fracture place, and time from injury to surgery (Table 2, p > 0.05). These results suggest that M2-type macrophages are more activated and recruited to the injury sites in patients with clavicle fracture and concomitant TBI.