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. 2013 Nov 18;8(11):e81341.
doi: 10.1371/journal.pone.0081341. eCollection 2013.

Complement C3 and C5 deficiency affects fracture healing

Christian Ehrnthaller  1 , Markus Huber-Lang, Per Nilsson, Ronny Bindl, Simon Redeker, Stefan Recknagel, Anna Rapp, Tom Mollnes, Michael Amling, Florian Gebhard, Anita Ignatius
Affiliations

Complement C3 and C5 deficiency affects fracture healing

Christian Ehrnthaller et al. PLoS One. .

Abstract

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3(-/-) and C5(-/-) as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3(-/-): -25%, p=0.02; C5(-/-): -20% p=0.052) and newly formed bone (C3(-/-): -38%, p=0.01; C5(-/-): -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3(-/-) mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Xray examination of the skeleton.
Whole-body Xrays of complement-deficient and corresponding wildtype mice immediately following surgery. The osteotomy of the right femur was stabilized using an external fixator, which was mounted with two mini-Schanz screws each in the proximal and distal bone fragment. A: C3-/-mouse; B: C3+/+ mouse; C: C5-/- mouse; D: C5+/+ mouse.
Figure 2
Figure 2. Analyzes of the growth plates.
A, D: Growth plate height in complement-deficient and corresponding wildtype mice. Results are displayed as mean ± 2×SEM; * = p≤0.05. B, C, E, F: Histological slides of the growth plates; B: C3+/+ mouse; C: C3-/- mouse; E: C5+/+ mouse; F: C5-/- mouse.
Figure 3
Figure 3. Complement serum analysis.
C3a and C5a concentration in the serum preoperatively (Pre-Op), 1 and 3 days after the osteotomy. Results are displayed as mean ± 2×SEM; * = p≤0.05; A: C3a in C3-/-; B: C5a in C3-/-; C: C3a in C5-/-; D: C5a in C5-/-.
Figure 4
Figure 4. Histological evaluation in C3-/-.
Histological evaluation of the fracture callus in the absence of C3. Area of the total callus and of cartilage, soft tissue and newly formed bone of the fracture callus of C3-/- and the corresponding wildtype mice. Results are displayed as mean ± 2×SEM; * = p≤0.05; A: day 7 after osteotomy; B: day 21 after osteotomy.
Figure 5
Figure 5. Histology.
Histological slides of the fracture callus of complement-deficient and corresponding wildtype mice 7 and 21 days after osteotomy. Giemsa staining. Bar = 200 µm.
Figure 6
Figure 6. Immunostaining for IL-6.
Histological slides from all 4 experimental groups after immunostaining for IL-6. Displayed are sections from the periosteal callus with predominant staining of the hypertrophic chondrocytes. Scale for all slides 100 µm as indicated. C: cortical bone; *: proliferating osteoblasts and hypertrophic chondrocytes.
Figure 7
Figure 7. Results fracture healing in C3-/-.
Biomechanical and µCT analyses of the fracture callus in the absence of C3 at 21 days after the osteotomy. A: Bending stiffness of the fracture callus. B: Total callus volume. C: Bone volume/total volume. D: Maximal moment of inertia. Results are displayed as mean ± 2×SEM; * = p≤0.05.
Figure 8
Figure 8. Histological evaluation in C5-/-.
Histological evaluation of the fracture callus in the absence of C5. Area of the total callus and of cartilage, soft tissue and newly formed bone of the fracture callus of C5-/- and the corresponding wildtype mice. Results are displayed as mean ± 2×SEM; * = p≤0.05; A: day 7 after osteotomy; B: day 21 after osteotomy.
Figure 9
Figure 9. Results fracture healing in C5-/-.
Biomechanical and µCT analyses of the fracture callus in the absence of C5 at 21 days after the osteotomy. A: Bending stiffness of the fracture callus. B: Total callus volume. C: Bone volume/total volume. D: Maximal moment of inertia. Results are displayed as mean ± 2×SEM; * = p≤0.05.
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