Bone formation in interleukin‐4 and interleukin‐13 depleted mice

Figures & data

Table 1.  Protocol for animals (all male) in the fracture study

Mouse	Genotype	Killed at	Radiography	pQCT	Bio-mech.	Histology
1	KO	5 weeks	X	X		X^b
2c	KO	perop.
3	KO	5 weeks	X	X		X^b
4	WT	5 weeks	X		X	X
5	WT	5 weeks	X	X		X^b
6	WT	5 weeks	X	X		X^b
7	WT	5 weeks	X		X	X
8	WT	5 weeks	X	X	X
9	WT	5 weeks	X		X
10^c	KO	postop.
11	KO	5 weeks	X		X^a	X
12	WT	5 weeks	X	X	X
13	KO	5 weeks	X	X	X	X
14	WT	5 weeks	X	X	X	X
15	WT	5 weeks	X	X	X
16	WT	5 weeks	X	X	X
17	KO	5 weeks	X	X	X	X
18	WT	5 weeks	X		X	X
19	KO	5 weeks	X	X	X	X
20	WT	5 weeks	X	X	X	X
21	KO	5 weeks	X	X	X
22	KO	5 weeks	X	X	X	X
23 ^c	KO	postop.
24	KO	5 weeks	X	X	X	X

^a Broken at dissection and therefore excluded from biomechanical testing.

^b Selected for unbroken callus histology and therefore excluded from biomechanical testing.

^c Animals died peroperatively or postoperatively (within 24 h) for anesthesiological reasons.

Table 2.  Protocol for animals in the DXBM study

Animals	Genotype	Killed at^a	X-ray	Ash	Histol.
Male
1	KO	5 weeks	X	X	X
2	KO	5 weeks	X	X
3	KO	5 weeks	X	X
4	KO	postop.
5	KO	5 weeks	X	X	X
6	KO	5 weeks	X	X	X
7	KO	5 weeks	X	X	X
8	KO	5 weeks	X	X	X
9	KO	5 weeks	X	X	X
10	KO	5 weeks	X	X	X
11	KO	5 days
12	KO	5 weeks	X	X
13	WT	postop.
14	WT	postop.
15	WT	5 weeks	X	X	X
16	WT	5 weeks	X	X	X
17	WT	5 weeks	X	X	X
18	WT	postop.
19	WT	7 days
20	WT	5 weeks	X	X	X
21	WT	5 weeks	X	X	X
22	WT	5 weeks	X	X	X
23	WT	5 weeks	X	X
24	WT	5 weeks	X	X	X
Female
25	KO	postop.
26	KO	5 weeks	X	X	X
27	KO	5 weeks	X	X
28	KO	5 weeks	X	X	X
29	KO	5 weeks	X	X	X
30	KO	5 weeks	X	X	X
31	KO	5 weeks	X	X	X
32	KO	5 weeks	X	X	X
33	KO	5 weeks	X	X
34	KO	5 weeks	X	X	X
35	KO	5 weeks	X	X
36	KO	5 weeks	X	X
37	WT	5 weeks	X	X	X
38	WT	5 weeks	X	X
39	WT	5 weeks	X	X
40	WT	5 weeks	X	X	X
41	WT	5 weeks	X	X	X
42	WT	5 weeks	X	X
43	WT	5 weeks	X	X
44	WT	5 weeks	X	X
45	WT	5 weeks	X	X	X
46	WT	5 weeks	X	X	X
47	WT	5 weeks	X	X	X
48	WT	5 weeks	X	X

^a5 animals died peroperatively or postoperatively (within 24 h) for anesthesiological reasons. 2 animals died, on day 5 and day 7. Autopsy was inconclusive; however, bite injuries were suspected.

Figure 1. Faxitron image of fracture specimens, stabilized by intramedullary nail, at killing 5 weeks postoperatively. WT animal (left) and IL‐4-/-IL‐13-/‐animal (right).

Table 3.  Callus bone parameters in dissected femora 5 weeks after fracture. Measurement by 5 consecutive pQCT scans over the center of the callus in adult male WT (n = 8) and KO (n = 8) mice. Values are mean (SEM)

	Control	Knockout	p-value^a
Volume (mm^3)	3.5 (0.47)	2.9 (0.46)	> 0.1
BMD (mg/mm^3)	0.3 (0.01)	0.3 (0.01)	> 0.1
BMC (mg)	1 (0.12)	0.9 (0.11)	> 0.1

^aTwo‐tailed Student's t-test.

Table 4.  Mechanical strength of callus as measured by three‐point bending 5 weeks after femur fracture in adult male WT (n = 10) and KO (n = 6) mice. Values are mean (SEM)

	Control	Knockout	p‐value a
Load (N)	10 (0.94)	13 (2.4)	>0.1
Displacement (mm)	0.79 (0.08)	0.71 (0.07)>0	>0.1
Stiffness (N/mm)	14 (1.6)	20 (4.5)	>0.1
Energy at failure (J)	3.5 (0.5)	2.9 (0.3)	>0.1
^a Mann‐Whitney U-test.

Table 5.  Histology fracture model

	Male IL‐4/13 knockout mice (n = 8)					Male WT mice (n = 7)
	Callus	Marrow	Cortical	Periost.	Muscle	Callus	Marrow	Cortical	Periost.	Muscle
GAP‐43	−	+	−	+	+	−	+	−	+	+
PGP 9.5	−	−	−	+	+	−	−	−	+	+
CGRP	−	+		+	+	-			+	+
NPY	−	+	−	+	+	−	−	−	+	+
PECAM‐1	+	−	+	−	−	+	−	+	−	−
H&E	sparse					sparse

Histological analysis of bone formation (H&E), presence of nerve fibers (staining for GAP‐43, PGP 9.5, CGRP, NPY) and vascularity in and adjacent to fracture callus (at 5 weeks) for male IL‐4/13 knockout and WT mice. Gap‐43 staining indicates immature nerve fibers, PGP 9.5 mature nerve fibers, CGRP sensory nerve fibers, and NPY indicates autonomous nerve fibers. PECAM‐1 staining indicates vascular infiltration. Both biomechanically tested calluses (IL‐4/13 knockout: n = 6; WT: n = 5) and intact calluses (IL‐4/13 knockout: n = 2; WT: n = 2) are included in the table.

Figure 2. Faxitron image of DXBM implant specimens at killing 5 weeks postoperatively. WT animal (left) and IL‐4-/-IL‐13-/‐animal (right).

Figure 3. Ash weight of implants in the 2 groups (WT and IL‐4-/-IL‐13-/-) at killing 5 weeks after implantation. Values are given as mean ± SEM. * p < 0.05, ** p < 0.01 relative to control (Student t-test).

Table 6.  Histology DXBM model

	IL‐4/1E knockout mice (M 7 /F7)				WT mice (M 7 / F 6)
	Impl M/F	Marrow M/F	Capsule M/F	Muscle M/F	Impl M/F	Marrow M/F	Capsule M/F	Muscle M/F
GAP	-/-	-/-	+ / +	+ / +	-/-	-/-	+ / +	+ / +
PGP	-/-	-/-	+ / +	+ / +	-/-	-/-	+ / +	+ / +
CGRP	-/-	-/-	-/-	+ / +	-/-	-/-	+ /-	+ / +
NPY	-/-	-/-	-/-	+ / +	-/-	-/-	+ / +	+ / +
PECAM‐1	-/-	+ / +	+ / +	+ / +	+ / +	+/ +	+ / +	+ / +
H&E		bone in 13	out of 14		i	bone in 1	3 out of 13

Histological analysis of bone formation (H&E), presence of nerve fibers (staining for GAP‐43, PGP 9.5, CGRP, NPY) and vascularity in and adjacent to DBMX implants (at 5 weeks) for male and female IL‐4/13 knockout and WT mice. Gap‐43 staining indicates immature nerve fibers, PGP 9.5 mature nerve fibers, CGRP sensory nerve fibers, and NPY indicates autonomous nerve fibers. PECAM‐1 staining indicates vascular infiltration.

Figure 4. Vascularity in and adjacent to DXBM implant in male WT mouse as shown by staining for platelet endothelial cell adhesion molecule‐1 (PECAM‐1/CD31). Mu: muscle; C: capsule; I: implant; and Ma: marrow. Arrows indicate blood vessels.

Figure 5. Autonomic nerve fibers (NPY) in the capsule of a WT female mouse, shown by arrows. Mu: muscle; C: capsule; and I: implant.