The expressions of NGF and VEGF in the fracture tissues are closely associated with accelerated clavicle fracture healing in patients with traumatic brain injury

Figures & data

Figure 1 Fracture healing time (A) and callus volume (B) in clavicle fracture alone group and clavicle fracture and concomitant TBI group were based on clinical and radiological examinations at 1, 2, 4, 8, 12, 16, 20, and 24 weeks after trauma.

Abbreviations: CF, clavicle fracture alone; CFT, clavicle fracture and concomitant TBI; TBI, traumatic brain injury.

Figure 2 The representative, longitudinal chest radiographs of the clavicle fracture alone group (upper panels) and clavicle fracture and concomitant TBI group (lower panels) were collected at the time points of pre-operation, 4 and 12 weeks post operation from the same patients.

Note: The blue arrows indicate the fracture lines.
Abbreviations: CF, clavicle fracture alone; CFT, clavicle fracture and concomitant TBI; TBI, traumatic brain injury.

Table 1 The demographics of clinical profile of patients in CF and CFT groups

Features	CF group	CFT group	P-value
Age (years)	36.0±15.3	34.0±15.2	0.657
Gender			0.820*
Male	21 (84.0)	19 (86.4)
Female	4 (16.0)	3 (15.6)
GCS	15±0	11.5±1.74	0.000
Time from injury to surgery			0.706*
≤7 days	10 (40.0)	10 (45.5%)
8–14 days	15 (60.0)	12 (54.5%)
Callus volume (cm^3)	8.53±2.78	21.95±10.30	0.000#
Density of NG F^+ cells (Num/0.01 mm^2)	69.80±12.31	178.00±31.33
Density of VEG F^+ cells (Num/0.01 mm^2)	27.68±7.15	97.55±13.45	0.000#
Density of CD31^+ cells (positive area/total field area)	2.19±1.38	5.69±1.87	0.000

Notes: Data are shown as mean±SD or n/N (%).

* Chi-squared test was used for the analysis.

# Mann–Whitney Test was used for the analysis. Student’s t-test was used in all other analysis.

Abbreviations: CF, clavicle fracture alone; CFT, clavicle fracture and concomitant traumatic brain injury; GCS, Glasgow Coma Score; NGF, nerve growth factor; Num, the number of positive cells; VEGF, vascular endothelial growth factor.

Figure 3 The concentrations of serum NGF in patients with clavicle fracture alone (CF group) and in patients with clavicle fracture and concomitant traumatic brain injury (CFT group) were measured at 1, 7, 14, and 21 days after surgery.

Note: Two-way repeated measures ANOVA analysis was followed by Bonferroni post-hoc test.
Abbreviations: CF, clavicle fracture alone; CFT, clavicle fracture and concomitant traumatic brain injury; NGF, nerve growth factor.

Figure 4 Expression of NGF, VEGF, and CD31 in callus tissue of clavicle fracture.

Notes: The representative immunohistochemistry images (A) showed the expression of NGF, VEGF, and CD31 in callus tissue of clavicle fracture alone (CF group) and clavicle fracture and concomitant TBI (CFT group). The tissue was collected during the surgery which was conducted between 8 and 14 days after injury; scale bars set at 50 µm. The density of CD31 (B), NGF (C) and VEGF (D) positive cells in CF and CFT groups were analyzed with the software of Image-pro plus 6.0.
Abbreviations: CF, clavicle fracture alone; CFT, clavicle fracture and concomitant TBI; NGF, nerve growth factor; TBI, traumatic brain injury; VEGF, vascular endothelial growth factor.

Table 2 The density of NGF⁺ cells in callus tissues is correlated with serum NGF levels (n=47)

	D1	D7	D14	D21
Density of NGF^+ cells
Pearson correlation	0.796*	0.827*	0.929*	0.814*
Significance (two-tailed)	0.000	0.000	0.000	0.000

Notes: D1, D7, D14, and D21: serum NGF concentrations at 1, 7, 14, and 21 days after surgery, respectively.

* Correlation is significant at the level of P<0.01 (two-tailed).

Abbreviation: NGF, nerve growth factor.

Table 3 Partial correlations among healing time and clinical variables by using surgery time post injury as control variable (n=47)

		Healing time	Age (years)	Gender	GCS	Injury type	Callus volume (cm^3)	Density of NGF^+ cells (Num/0.01 mm^2)	Density of VEGF^+ cells	Density of CD31^+ cells (positive area/total field area)
Healing time (days)	r	1	0.110	−0.047	0.765**	−0.747**	−0.632**	−0.837**	−0.831**	−0.847**
	P		0.467	0.757	0.000	0.000	0.000	0.000	0.000	0.000
Age (years)	r	0.110	1	0.049	0.089	−0.066	0.043	−0.052	−0.134	−0.057
	P	0.467		0.748	0.558	0.662	0.777	0.729	0.373	0.705
Gender	r	−0.047	0.049	1	−0.043	0.040	0.075	0.063	0.089	0.168
	P	0.753	0.748		0.778	0.790	0.622	0.677	0.557	0.264
GCS	r	0.765**	0.089	−0.043	1	−0.884**	−0.819**	−0.932**	−0.901**	−0.785**
	P	0.000	0.558	0.778		0.000	0.000	0.000	0.000	0.000
Injury type	r	−0.747**	−0.066	0.040	−0.884**	1	0.708**	0.951**	0.966**	0.813**
	P	0.000	0.662	0.790	0.000		0.000	0.000	0.000	0.000
Callus volume (cm^3)	r	−0.632**	0.043	0.075	−0.819**	0.708**	1	0.774**	0.726**	0.677**
	P	0.000	0.777	0.622	0.000	0.000		0.000	0.000	0.000
Density of NGF^+ cells (Num/0.01 mm^2)	r	−0.837**	−0.052	0.063	−0.932**	0.951**	0.774**	1	0.972**	0.879**
	P	0.000	0.729	0.677	0.000	0.000	0.000		0.000	0.000
Density of VEGF^+ cells (Num/0.01 mm^2)	r	–0.831**	−0.134	0.089	−0.901**	0.966**	0.726**	0.972**	1	0.864**
	P	0.000	0.373	0.557	0.000	0.000	0.000	0.000		0.000
Density of CD31^+ cells (positive area/total field area)	r	–0.847**	−0.057	0.168	−0.785**	0.785**	0.677**	0.879**	0.864**	1
	P	0.000	0.705	0.264	0.000	0.000	0.000	0.000	0.000

Notes:

** Correlation is significant at the level of P<0.01 (two-tailed).

Abbreviations: GCS, Glasgow Coma Scale; NGF, nerve growth factor; VEGF, vascular endothelial growth factor.