The anaphylatoxin receptor C5aR is present during fracture healing in rats and mediates osteoblast migration in vitro

Abstract

Background: There is evidence that complement components regulate cytokine production in osteoblastic cells, induce cell migration in mesenchymal stem cells, and play a regulatory role in normal enchondral bone formation. We proved the hypothesis that complement might be involved in bone healing after fracture.

Methods: We investigated the expression of the key anaphylatoxin receptor C5aR during fracture healing in rats by immunostaining after 1, 3, 7, 14, and 28 days. C5aR expression was additionally analyzed in human mesenchymal stem cells (hMSC) during osteogenic differentiation, in human primary osteoblasts, and osteoclasts by reverse transcriptase polymerase chain reaction and immunostaining. Receptor functionality was proven by the migratory response of cells to C5a in a Boyden chamber.

Results: C5aR was expressed in a distinct spatial and temporal pattern in the fracture callus by differentiated osteoblast, chondroblast-like cells in cartilaginous regions, and osteoclasts. In vitro C5aR was expressed by osteoblasts, osteoclasts, and hMSC undergoing osteogenic differentiation. C5aR was barely expressed by undifferentiated hMSC but was significantly induced after osteogenic differentiation. C5aR activation by C5a induced strong chemotactic activity in osteoblasts, and in hMSC, which had undergone osteogenic differentiation, being abolished by a specific C5aR antagonist. In hMSC, C5a induced less migration reflecting their low level of C5aR expression.

Conclusions: Our in vitro and in vivo results demonstrated the presence of C5aR in bone forming osteoblasts and bone resorbing osteoclasts. It is suggested that C5aR might play a regulatory role in fracture healing in intramembranous and in enchondral ossification, one possible function being the regulation of cell recruitment.

Figure 1

C5aR mRNA expression relative to the house keeping gene GADPH; o-hMSC, cells cultivated under osteogenic conditions for 14 days, 21 days, and 28 days; OB, primary osteoblasts; six independent experiments, which were performed in triplicates. *p ≤ 0.05.

Figure 2

Proof of successful osteogenic differentiation by von Kossa (left side) and alkaline phosphatase staining (right side); negative von Kossa (A) and alkaline phosphatase (B) staining of undifferentiated hMSC; positive von Kossa (C) and alkaline phosphatase (D) staining of hMSC cultivated under osteogenic conditions for 21 days; positive von Kossa (E) and alkaline phosphatase (F) staining of hMSC cultivated under osteogenic conditions for 21 days and treated with 100 ng/ml C5a.

Figure 3

Immunostaining of C5aR of bone cells in vitro; (A) C5aR negative undifferentiated hMSC; (B) negative control after incubation with goat IgG; (C) C5aR positive differentiated hMSC; (D) C5aR positive primary osteoblasts; (E) C5aR positive multinucleated osteoclast; (F) corresponding negative control after Incubation with goat IgG, multiple cell nuclei were counterstained with hematoxylin.

Figure 4

C5a-induced migration of undifferentiated hMSC (white columns) and primary osteoblasts (grey columns). C, basal conditions without addition of C5a. Three independent experiments, which were performed in quadruplicates. *p ≤ 0.05.

Figure 5

Left side, checkerboard analysis, 100 ng/ml C5a was added to the lower well alone or to both the lower and the upper wells of a Boyden chamber; only directed cell migration occurred. Right side, C5a induced migration could be completely abolished by preincubation with a C5aR specific antagonist (Rec-Inhib). Osteoblasts from one donor, experiments were performed in quadruplicates. *p ≤ 0.05.

Figure 6

Lines 1–4, Immunostaining of C5aR in the fracture callus of the rat tibia during the time course of fracture healing from day 3 until day 28. Left side, Overviews of the fracture callus on specified days during healing (Giemsa staining). (A-H) Detailed pictures. (A) Inflammatory cells at the fracture gap positively stained for C5aR. (B) Periosteum with osteoblasts positively stained for C5aR. (C) Chondroblast-like cells in the fracture callus positively stained for C5aR. (D) Osteoblasts in newly formed bone positively stained for C5aR. (E) Positively stained osteocytes in cortical bone. (F) Newly formed bone with osteocytes and osteoblasts positively stained for C5aR. (G) Chondroblast-like cells in the fracture callus positively stained for C5aR. (H) Positively stained cells in the fracture region. Line 5, Immunostaining of C5aR in a healthy human bone. Left side: overview. (I and J) Detailed pictures. (I) Osteocytes positively stained for C5aR. (J) Osteoblasts at the surface of a trabecular bone positively stained for C5aR.