Chondrocyte TUNEL staining is reduced in GIT1 KO mice.

<p>Chondrocyte apoptosis was assessed in WT and GIT1 KO mice at 14 and 21 days post-fracture. Representative TUNEL immunofluorescence and DAPI staining at both time points is presented in WT mice (A/C and E/G respectively) and in GIT1 KO mice (B/D and F/H respectively). Quantitative histomorphometric analyses of the number of TUNEL-positive cells per unit area in triplicate sections from three WT and GIT1 KO mice at 14 and 21 days post-fracture are presented (I). Bars represent the percent of TUNEL positive cells/mm²+/− SEM (*p<0.05, N = 3).</p

Fracture callus vascularity is reduced in GIT1 KO mice.

<p>To visualize and quantify callus vascularity, WT and GIT1 KO mice were perfused with lead chromate microfilm perfusion reagent. Harvested femora were decalcified and representative vascular microCT reconstructions from each experimental group at 7, 14 and 21 days post-fracture are presented. Reduced vascularity in GIT1 KO mice (A, C, E) compared to WT control mice (B, D, F) was evident at all time points. Quantification of callus vascular parameters, including Vessel Volume (G), Vessel Number (H), Vessel Spacing (I) and Connection Density (J) supported these findings, with GIT1 KO mice possessing reduced callus vessel volume, vessel number and connection density and increased space between vessels compared to callus from WT mice. Bars represent mean for each value +/− SEM (N = 3, *p<0.05).</p

PECAM1⁺ blood vessel number is reduced in GIT1 KO mice.

<p>Representative PECAM1 immunofluorescence is presented at 7 and 14 days post-fracture in WT mice (A and C) and GIT1^−/− mice (B and D). Histomorphometry was performed to quantify the average number of positively-stained blood vessels present in each field of view on each section analyzed. Three sections (from 3 levels within each callus, 25–50 µm apart) were viewed using the 10× objective, with counts being collected from 3 fields of view in each section. All counts from each callus (9 fields total) were averaged. Vessel counting using this approach confirmed the immunofluorescence in panels A–D, with WT calluses possessing between 2 and 3-fold more PECAM1⁺ vessels than GIT1 KO calluses at both time points (E). Bars represent mean number of PECAM1⁺ vessels/field +/− SEM (*p<0.01, N = 3).</p

Type 2 collagen-containing matrix persists in GIT1 KO mice.

<p>Tissue sections cut from WT and GIT1 KO mice were analyzed for COL2A1 content using an immunohistochemistry approach. Representative stains at 7, 14 and 21 days post-fracture are depicted, with asterisks denoting areas within the callus at 2 and 3 weeks post-fracture in GIT1 KO mice (D and F respectively) that have more robust/persistent staining.</p

Disjunction persists at 14 days post-fracture in GIT1 KO mice.

<p>Femur fractures were induced in 10-week-old WT and GIT1 KO mice. Fractured femora were harvested for analysis at 7, 14, and 21 days post-injury. Radiographs obtained at the 14 day time point consistently revealed radiolucency in GIT1 KO calluses (B, red arrow) compared with calluses from WT mice (A, yellow arrow). This was supported by microCT analysis, which revealed lack of bridging mineral in GIT1 KOs (D, red arrows) compared to a connected shell of mineral in WT controls (C). Further quantification of callus geometry via microCT indicated that there were no differences in mineralized callus volume between WT and GIT1 KO mice (E). Bars represent mean callus volume (mm³) +/− SEM (N = 3, *p<0.05).</p

Chondrocyte proliferation is reduced in GIT1 KO mice.

<p>Representative PCNA staining is shown at 7 and 14 days post-fracture in WT mice (A and C) and at 7 (B) and 14 days (D) post-fracture in GIT1 KO mice (B and D respectively). Histomorphometry was performed on triplicate sections from multiple mice to quantify the number of PCNA-positive cells per unit callus area (E). The data is presented as mean of the number of PCNA positive cells/mm²+/− SEM (*p<0.05, N = 3).</p

Osteoclast number is reduced in GIT1 KO mice.

<p>The presence of osteoclasts in the fracture callus of WT and GIT1 KO mice was assessed at 7 (A and B respectively), 14 (C and D respectively) and 21 days (E and F respectively) post-fracture via TRAP staining. Histomorphometry to quantify percentage of osteoclast surface (G) and osteoclast number per unit bone surface (H) was also performed on triplicate sections from multiple mice, with bars representing the mean for each parameter +/− SEM (*p<0.05, N = 3).</p

Table1_A prospective marker for the prediction of postoperative deep venous thrombosis: Neutrophil extracellular traps.XLSX

Deep venous thrombosis (DVT) is a common medical complication in patients with lumbar fractures. The current study aimed to investigate the predictive value of neutrophil extracellular traps (NETs) in postoperative DVT formation in patients with lumbar fractures and to develop a nomogram relating clinical admission information for prediction. Patients who underwent open reduction and pedicle screw internal fixation in the treatment of single-segment lumbar fracture in the Department of Spine Surgery, the First Affiliated Hospital of Nanjing Medical University, from December 2020 to June 2022 were enrolled in this study. Baseline data and laboratory results were collected from enrollees, and the primary study endpoint event was the occurrence of DVT in patients after surgery. Multivariable logistic regression analysis was used to identify risk factors associated with higher odds of DVT after surgery. A nomogram was constructed using the results of the multivariable model. The calibration plot and receiver operating characteristics (ROC) curve were used to show the satisfactory predictive capacity of the model. Of these 393 patients who did not have DVT preoperatively, 79 patients developed it postoperatively, and 314 did not, respectively. Multivariate analysis showed that higher body mass index (BMI) (BMI between 24 and 28: RR = 1.661, 95% CI = 0.891–3.094; BMI ≤28: RR = 5.625, 95% CI = 2.590–12.217; reference: BMI <24), neutrophils (RR = 1.157, 95% CI 1.042–1.285), D-dimer (RR = 1.098, 95% CI 1.000–1.206), and citrullinated histone H3 (CitH3) (RR = 1.043, 95% CI 1.026–1.060) were independent risk factors for postoperative DVT. Using the multivariable analysis, we then constructed a nomogram to predict DVT, which was found to have an area under the curve of 0.757 (95% CI = 0.693–0.820). Calibration plots also showed the satisfied discrimination and calibration of the nomogram. In conclusion, patients with lumbar fractures with postoperative DVT had higher levels of NETs in the circulation preoperatively compared to those without postoperative DVT. Furthermore, based on BMI, D-dimer, neutrophils, and CitH3, we developed a predictive model to predict postoperative DVT incidence in these patients.</p