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RESEARCH ARTICLE
Osteoblast-specific overexpression of
complement receptor C5aR1 impairs fracture
healing
Stephanie Bergdolt
1
,Anna Kovtun
1
,Yvonne Ha
¨gele
1
,Astrid Liedert
1
,Thorsten Schinke
2
,
Michael Amling
2
,Markus Huber-Lang
3
,Anita Ignatius
1
*
1Institute of OrthopedicResearch and Biomechanics, University of Ulm, Ulm, Germany, 2Department of
Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany,
3Institute of Clinical and Experimental Traumaimmunology, University of Ulm, Ulm, Germany
*anita.ignatius@uni-ulm.de
Abstract
The anaphylatoxin receptor C5aR1 plays an important role not only in innate immune
responses, but also in bone metabolism and fracture healing, being highly expressed on
immune and bone cells, including osteoblasts and osteoclasts. C5aR1 induces osteoblast
migration, cytokine generation and osteoclastogenesis, however, the exact role of C5aR1-
mediated signaling in osteoblasts is not entirely known. Therefore, we hypothesized that
osteoblasts are essential target cells for C5a and that fracture healing should be disturbed
in mice with an osteoblast-specific C5aR1 overexpression (Col1a1-C5aR1). Osteoblast
activity in vitro,bone phenotype and fracture healing after isolated osteotomy and after com-
bined osteotomy with additional thoracic trauma were analyzed. The systemic and local
inflammatory reactions were analyzed by determining C5a and IL-6 concentrations in blood,
bronchoalveolar lavage fluid and fracture callus and the recruitment of immune cells. In
vitro,osteoblast proliferation and differentiation were similar to wildtype cells, and phosphor-
ylation of p38 and expression of IL-6 and RANKL were increased in osteoblasts derived
from Col1a1-C5aR1 mice. Bone phenotype and the inflammatory reaction were unaffected
in Col1a1-C5aR1 mice. Fracture healing was significantly impaired as demonstrated by sig-
nificantly reduced bone content, bone mineral density and flexural rigidity, possibly due to
significantly increased osteoclast numbers. C5aR1 signaling in osteoblasts might possibly
affect RANKL/OPG balance, leading to increased bone resorption. Additional trauma signifi-
cantly impaired fracture healing, particularly in Col1a1-C5aR1 mice. In conclusion, the data
indicate that C5aR1 signaling in osteoblasts plays adetrimentalrole in bone regeneration
after fracture.
Introduction
In recent years, evidence has accumulated that the complement system, an essential part of
innate immunity, is involved in the regulation of bone turnover and in the pathogenesis of
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 1 / 17
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OPEN ACCESS
Citation: Bergdolt S, Kovtun A, Ha¨gele Y, Liedert A,
Schinke T, Amling M, et al. (2017) Osteoblast-
specific overexpression of complement receptor
C5aR1 impairs fracture healing. PLoS ONE 12(6):
e0179512. https://doi.org/10.1371/journal.
pone.0179512
Editor: Jose Manuel Garcia Aznar, University of
Zaragoza, SPAIN
Received: February 17, 2017
Accepted: May 30, 2017
Published: June 14, 2017
Copyright: ©2017 Bergdolt et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License,which
permits unrestricteduse, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data AvailabilityStatement: All relevant data are
within the paper.
Funding: The study was funded by the German
Research Foundation (DFG) in the framework of
the Clinical Research Group KFO200 (IG18/14-2)
and the Collaborative Research Center CRC1149
(INST 40/491-1). The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests:The authors have declared
that no competing interests exist.
bone disorders, including fracture healing. However, the underlying mechanisms are currently
poorly understood [1–3].
The complement system, anetwork of more than 30 soluble and membrane-bound mole-
cules, serves as apowerful danger-sensing system, which is activated by exogenous and en-
dogenous danger molecules [4]. It can be activated intrinsically by the alternative, lectin or
classical pathway or extrinsically by serine proteases, including thrombin of the coagulation
system [4,5]. All pathways lead to the generation of the anaphylatoxin C5a, which crucially
triggers the inflammatory response of immune cells. For example, C5a induces the migration
of inflammatory cells, the degranulation of mast cells and the release of inflammatory cyto-
kines and regulates apoptosis [6–8]. The pro-inflammatory actions of C5a are mediated by its
receptor C5aR1, aclassical Gprotein-coupled receptor, expressed on the plasma membrane of
avariety of cells, particularly immune cells, including neutrophils, eosinophils and basophils
[6,9]. However, C5aR1 is also expressed on non-immune cells, including, among others, endo-
thelial cells and neurons, suggesting that complement-mediated signaling is not limited to
inflammatory cells [8]. We and others have demonstrated that C5aR1 is also strongly upregu-
lated during osteogenic differentiation of mesenchymal stem cells (MSCs) and induced osteo-
blast migration and the production of pro-inflammatory cytokines, including interleukin (IL)-
6and IL-8 in vitro [1,10,11]. Notably, C5a also enhanced the expression of receptor activator
of nuclear factor-κBligand (RANKL) in osteoblasts, akey stimulator of osteoclastogenesis,
indicating that C5a may indirectly induce osteoclastic bone resorption [12]. C5aR1 is also
expressed on osteoclasts and can directly regulate their differentiation and activity [1,13].
Taken together, these data indicate that the activation of the C5a/C5aR1 axis could modulate
bone cell activity and their immune-modulatory response [3]. The C5aR1 axis in bone cells
might be particularly important under inflammatory conditions after bone injury. Accord-
ingly, we demonstrated that C5aR1 was strongly upregulated in the periosteum, where precur-
sor cells already start to proliferate and undergo osteogenic differentiation during the early
phase after bone fracture [1]. These osteoblasts may modulate the local immune response after
stimulation with C5a, which is locally and, depending on trauma severity, systemically gener-
ated as aresponse to damage-associated molecular patterns. Furthermore, C5aR1 was strongly
expressed by bone-forming osteoblasts in the fracture callus over the entire healing period [1],
indicating that osteoblasts may serve as target cells for C5a not only in the early inflammatory
phase but also later during bone repair. Confirming this, C5-deficient mice displayed dis-
turbed bone formation in the fracture callus [2]. However, the specific role of the C5a-
C5aR1-interaction in osteoblasts during bone healing is currently poorly understood.
Therefore, we investigated the function of the C5a/C5aR1 axis in osteoblasts during bone
repair using transgenic mice with an osteoblast-specific C5aR1 overexpression. We evaluated
fracture healing in amodel of uncomplicated isolated fracture and in amodel of impaired
healing by combining the fracture with an additional thoracic trauma, which is associated with
local and systemic complement activation and severe inflammation [14,15]. We hypothesize
that if osteoblasts were important target cells for C5a, fracture healing would be disturbed.
Materials and methods
Mouse model
The mouse experiments were performed according to the international regulations for the
care and use of laboratory animals and approved by the Local Ethical Committee (Regierung-
spra¨sidium Tu¨bingen, GER, Reg.Nr. 1096). All mice were housed in groups of up to four mice
per cage with a14 hlight and 10 hdark cycle and received astandard mouse feed (ssniff
1
R/
M-H, V1535-300, Ssniff, Soest, Germany) and water ad libitum.Mice with an osteoblast-
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 2 / 17
specific overexpression of C5aR1 (Col1a1-C5aR1) were generated as follows: The open reading
frame encoding C5aR1 was placed under the control of the osteoblast-specific 2.3 kb Col1a1
promotor fragment. To confirm C5aR1 overexpression, Col1a1-C5aR1 mice were genotyped
using the forward primer 5-ACTACATCCTGGTCATCATCCTGC-3 and the reverse primer
5-CCAGCAGGAAACGGTCGGCA-3.Wildtype littermates (WT) were used as controls.
Cultivation of primary mouse osteoblasts
Primary osteoblasts were isolated from long bones of 8- to 12-week-old WT and Col1a1--
C5aR1 mice. Briefly, long bones were harvested and bone marrow was flushed out using phos-
phate-buffered saline (PBS, Biochrom, Berlin, Germany). The diaphyses were cut into small
pieces, which were digested with collagenase (1 mg/ml collagenase type II (125 U/mg, Sigma-
Aldrich, Taufkirchen, Germany)) for 2 h at 37˚C and then washed twice with PBS. For osteo-
blast expansion, bone fragments were cultivated in culture medium (α-MEM, Biochrom) sup-
plemented with 10% heat-inactivated fetal calf serum (Gibco, Darmstadt, Germany), 100 U/ml
penicillin/streptomycin (Gibco), 1% L-glutamine (PAN-Biotech, Aidenbach, Germany) and
0.5% Fungizone
TM
(Amphotericin B, Gibco) at 37˚C and 5% CO
2
atmosphere.
To induce osteogenic differentiation, osteoblasts were seeded in 24-well plates (CELL-
STAR1,Greiner Bio-One) at adensity of 0.5 ×10
4
cells/cm
2
in the presence of 0.2 mM ascor-
bate-2-phosphate (Sigma-Aldrich) and 10 mM β-glycerophosphate (Sigma-Aldrich) for 14 d.
Alkaline-phosphatase staining (Sigma-Aldrich) was performed to confirm osteogenic differen-
tiation. To investigate whether the differentiation capacity was influenced by C5a, 0.1 μg/ml of
recombinant mouse C5a (R&D Systems, Minneapolis, USA) was added to the differentiation
medium. To investigate whether C5a can induce alocal inflammatory response in osteoblasts
after osteogenic differentiation, cells were stimulated with 0.1 μg/ml C5a for 6or 24 hand ana-
lyzed by western blot and real-time quantitative PCR (RT-qPCR), respectively.
Osteoblast proliferation was assessed using the BrdU Cell Proliferation Kit (Cell Signaling
Technology, Danvers, MA, USA) according to manufacturer’s recommendations. Briefly, oste-
oblasts were seeded in 96-well plates at adensity of 1.6 ×10
4
cells/cm
2
.BrdU detection anti-
body (1:100) was added to culture medium for 24 hat 37˚C. Cells were then incubated with a
horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT, before adding
3,3,5,5-Tetramethylbenzidin substrate for 25 min at RT. The cells were analyzed by spectro-
photometry at 450 nm.
Reverse transcription and real-time quantitative PCR
Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany)
according to the manufacturer’s instructions and cDNA was transcribed from 1μgRNA using
the Omniscript RT Kit (Qiagen). The StepOnePlus
TM
Real-Time PCR System (Applied Bio-
systems, Darmstadt, Germany) and the Platinum
1
SYBR
1
Green qPCR SuperMix-UDG
(Invitrogen ThermoFisher Scientific, Waltham, MA, USA) were used for cDNA amplification
and quantification according to the manufacturer’s instructions. Samples were initially incu-
bated at 50˚C and at 95˚C, for 2min each, followed by 40 cycles with the following cycling
conditions: 95˚C for 15 sand 60˚C for 1min. The amplification protocol was terminated
with amelting-curve step at 95˚C for 15 s, followed by 60˚C for 1min. Gene expression was
analyzed relative to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) using the ΔΔC
t
method with PCR-efficiency correction using LinRegPCR software
as described previously [16]. All primers were purchased from Invitrogen or Thermo Electron
(Ulm, Germany) (Table 1).
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 3 / 17
Western blot
Osteoblasts were lysed in Pierce1RIPA buffer (ThermoFisher Scientific, Waltham, MA,
USA), containing protease and phosphatase inhibitors (ThermoFisher Scientific). We resolved
10 μgof total protein on a10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
and transferred it to anitrocellulose membrane (Amersham
TM
Protran
TM
0.2 mNC, GE
Healthcare, Chicago, IL, USA). Antibody blocking was performed in Tris-buffered saline with
Tween20 (TBS-T) with 3% bovine serum albumin for 1 h at room temperature (RT). Primary
rat anti-mouse C5aR (CD88) (MA1-81761, 1:1000, ThermoFisher Scientific), rabbit anti-
mouse p-Erk1/2 (4370P, 1:1000, Cell Signaling Technology), rabbit anti-mouse Erk1/2 (4695P,
1:1000, Cell Signaling Technology), rabbit anti-mouse p-p38 (4511P, 1:1000, Cell Signaling
Technology), rabbit anti-mouse p38 (8690P, 1:1000, Cell Signaling Technology) and arabbit
anti-mouse GAPDH antibody (2118, 1:2000, Cell Signaling) were incubated overnight at
4˚C, followed by incubation with asecondary HRP-coupled anti-rat or anti-rabbit antibody
(1:15,000, Cell Signaling Technology), respectively, at RT for 1h. WesternBright
TM
Quantum
or WesternBright
TM
ECL chemiluminescent HRP substrate (both Advansta, CA, USA) were
added to the membranes for 2min at RT and the luminescent signal was captured using the
Fusion Molecular Imaging System (Vilber Lourmat, Eberhardzell, Germany) or by membrane
exposure to X-ray film.
Animal studies
To investigate whether osteoblast-specific C5aR1 overexpression influences bone development
and bone mass, the skeleton of 12- and 55-week-old male WT and Col1a1-C5aR1 mice was
analyzed by micro-computed tomography (μCT) and histomorphometry as described below.
Mice received subcutaneous injections of alizarin red and calcein green (both 30 mg/kg) (both
Sigma, Steinheim, Germany) 3and 12 days before bone harvesting to determine dynamic
bone formation.
To study fracture healing, 12-week-old male Col1a1-C5aR1 mice and WT littermates
received astandardized femur osteotomy as described previously [17]. Briefly, mice were anes-
thetized with 2% isoflurane (Forene, Abbott, Wiesbaden, Germany) and an external fixator
(axial stiffness 3N/mm, RISystem Ind., Davos, Switzerland) was attached to the femur using
four Schanz-screws. An osteotomy gap was created between the two inner pins using awire
saw of 0.4 mm diameter (RISystem Ind.). Immediately after the osteotomy, half of the mice
received an additional thoracic trauma to induce abilateral, isolated lung contusion via a
Table 1. Primer sequences.
Target Gene Forward Primer Sequence (5’–3’) Reverse Primer Sequence (5’–3’)
AP (Alpl) GCT GAT CAT TCC CAC GTT TT GAG CCA GAC CAA AGA TGG AG
OCN (Bglap) GCG CTC TGT CTC TCT GAC CT ACC TTA TTG CCC TCC TGC TT
BSP (Ibsp) GAA GCA GGT GCA GAA GGA AC GAA ACC CGT TCA GAA GGA CA
GAPDH (Gapdh) ACC CAG AAG ACT GTG GAT GG GGA TGC AGG GAT GAT GTT CT
IL-6 (Il6)TCC TTC CTA CCC CAA TTT CC GCC ACT CCT TCT GTG ACT CC
RANKL (Tnfsf11) ATC ATG AAA CAT CGG GAA GC CTT GGG ATT TTG ATG CTG GT
OPG (Tnfrsf11b) CTG CCT GGG AAG AAG ATC AG GCT CGA TTT GCA GGT CTT TC
Protein name with gene name in bracket. AP: alkaline phosphatase, OCN: bone gamma carboxyglutamate
protein/osteocalcin, BSP: bone sialoprotein, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, IL-6:
interleukin 6, RANKL: receptor activator of nuclear factor-κB ligand, OPG: osteoprotegerin.
https://doi.org/10.1371/journal.pone.0179512.t001
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 4 / 17
blast-wave generator centered 2cm above the middle of the thorax as described previously
[14,18]. For pain medication, all mice received 25 mg/l tramadol hydrochloride (Tramal1,
Gruenenthal GmbH, Aachen, Germany) in the drinking water 1 d before and up to 3 d after
surgery. Mice were sacrificed 3h, 1d, 3d, 14 d, 21 dand 25 dafter surgery using an overdose
of CO
2
and subsequent heart puncture with blood withdrawal. Blood for serum/plasma,
bronchoalveolar lavage (BAL) fluid and femur were harvested for further analyses.
Analysis of inflammatory mediators in blood, BAL fluids and callus
homogenates
Blood was harvested 3 h after surgery in microvettes (Sarstedt AG &Co., Nu¨mbrecht, Ger-
many) and centrifuged at 10,000 ×gfor 10 min for plasma collection or at 2800 ×gfor 10 min
for serum collection and stored at −80˚C. At the same time point, BAL fluid was collected by
flushing the lungs with 500 μlof ice-cold PBS. After centrifugation at 4000 ×gfor 10 min, the
supernatant was stored at −80˚C [19]. Fractured femurs were explanted 3 h after surgery,
shock-frozen in liquid nitrogen and stored at −80˚C. The fracture hematoma was collected in
150 μllysis buffer (10 mM Tris pH 7.5, 10 mM NaCl, 0.1 mM ethylenediaminetetraacetic acid,
0.5 mM Triton X-100, 0.02% NaN
3
,0.2 mM phenylmethylsulfonyl fluoride) with protease
inhibitors (1:100 vol/vol Sigma-Aldrich) and homogenized. After incubation for 30 min on
ice, the samples were centrifuged for 30 min at 10,000 ×gat 4˚C. The protein concentration in
the supernatant was determined using the Pierce
TM
BCA Protein Assay Kit (Fisher Scientific
GmbH, Schwerte, Germany) according to manufacturer’s protocol and lysates were stored at
−80˚C. C5a concentrations were determined in plasma, callus lysates and BAL fluid and IL-6
was determined in serum using mouse enzyme-linked immunosorbent assay (ELISA) kits
(C5a: R&D Systems, 1:100, 1:50 and 1:100; respectively; IL-6: BD Biosciences, Singapore, 1:2).
IL-6 concentrations in callus lysates and BAL fluid were determined using amouse Multiplex-
ELISA kit (Bio-Plex Pro Cytokine Assay, Bio-Rad, Hercules, CA, USA). Data were analyzed
using the standard curve of cytokine standards and values below the detection limit of the
assay were set to zero.
Micro-computed tomography
Following fixation in 4% formalin, the femurs were scanned by micro-computed tomography
(μCT) using aSkyscan 1172, (Kontich, Belgium) at 50 kV, 200 μAand aresolution of 8μm
[14]. Analysis of μCT data was performed according to the guidelines of Bouxsein and col-
leagues [20]and the standard American Society for Bone and Mineral Research (ASBMR)
guidelines for μCT analysis [21]. For bone phenotyping, the volumes of interest (VOIs) were
defined as follows: 168 μmheight in the mid-diaphysis of the cortical bone and for the trabecu-
lar bone in the distal part of the femur 200 μmabove the growth plate and 280 μmheight. For
analysis of fracture healing, the fracture gap was defined as the VOI. Global thresholding
(641.9 mg hydroxyapatite/cm
3
)was used to distinguish between mineralized and non-miner-
alized tissue to calculate tissue mineral density and trabecular thickness, number and separa-
tion (CTAnalyser, Skyscan) [22]. Bone mineral density was determined without thresholding.
Histology and immunohistochemistry
Femurs were fixed in 4% buffered formaldehyde and embedded in paraffin for Safranin-O
staining (14 dafter surgery) or in polymethylmethacrylate for Giemsa staining (21 dand 25 d
after surgery) and bone phenotyping. Longitudinal sections of the fractured femur were cut
in the anterior-posterior direction. Bone, cartilage and fibrous tissue areas were assessed
using image-analyzing software (MMAF Version 1.4.0 MetaMorph1,Leica, Heerbrugg,
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 5 / 17
Switzerland) and OsteoMeasure1image-analysis software (OsteoMetrics, Inc., GA, USA) was
used to determine the number of osteoblasts in toluidine blue-stained sections, the number of
osteoclasts in tartrate-resistant acid phosphatase (TRAP)-stained sections and the bone forma-
tion rate according to the guidelines of the ASBMR [21].
Sections of the fractured femurs were immunostained 1and 3 d after surgery for neutro-
phils (Ly-6G
+
,#127632, BioLegend, San Diego, CA, USA), macrophages (F4/80
+
,Bio-Rad
AbD Serotec GmbH, Puchheim, Germany) and T-lymphocytes (CD8
+
,AM11118PU-S, Acris,
Herford, Germany), and 14 dafter surgery for Runx2 (cs8486, Cell Signaling), osteocalcin
(orb77248, Biorbyt Ltd, Cambridge, UK), RANKL (sc-7628, Santa Cruz, Dellas, Texas, USA)
and osteoprotegerin (OPG, APO6631PU-N, Acris). After deparaffinizing the 4% buffered
formaldehyde-fixed femur sections in xylene, they were rehydrated and blocked (F4/80
+
,
Runx2 and osteocalcin with 5% goat serum; Ly6-G
+
,CD8
+
and OPG with 10% goat serum) for
1 h at RT. Sections were incubated with primary antibodies (Ly6-G
+
1:300, F4/80
+
1:500,
CD8
+
1:500, Runx2 1:50, osteocalcin 1:200, RANKL 1:100, OPG 1:400) at 4˚C overnight except
for CD8
+
for 2 h at RT and then all for 1 h at RT with respective secondary antibodies (goat
anti-rat or goat anti-rabbit, biotinylated, 1:200, Life Technologies, Carlsbad, CA, USA). To
detect the antibodies, ABC kit and NovaRED substrate (both Vector laboratories Inc., Burlin-
game, CA, USA) were applied according to the manufacturer’s protocol, followed by counter-
staining with hematoxylin. For analysis under alight microscope (Leica DMI6000 B, Leica,
Heerbrugg, Switzerland), the region of interest was defined as the periosteal callus between the
inner pins of the fixator. Macrophage staining was analyzed in the marrow cavity in direct
proximity to the fracture gap.
Biomechanical testing
Osteotomized and intact femurs were explanted 21 dand 25 dafter surgery and the flexural
rigidity was analyzed by anon-destructive, three-point bending test using amaterial testing
machine (Z10, Zwick GmbH, Ulm, Germany) [17]. Briefly, an axial bending load with aforce
of up to 4 N was applied on top of the callus or on the mid-point of the diaphysis of the intact
femur. The flexural rigidity (EI) was calculated from the slope of the force deflection curve.
The EI of the fractured femur was related to the contralateral femur of the same animal [17].
Statistical analysis
Proliferation and osteogenic differentiation of primary osteoblasts was analyzed in 5–6 inde-
pendent experiments. Bone phenotype and fracture healing were determined in 5–8 mice per
genotype and time point.
Groups were tested for normal distribution using the Shapiro-Wilk test and then compared
by either Kruskall-Wallis and Dunn’s post-hoc test or by one-way analysis of variance and Fish-
ers LSD post-hoc test using GraphPad Prism 6(GraphPad Software Inc., La Jolla, CA, USA).
Data are presented as mean ±standard deviation. The level of significance was set at p<0.05.
Results
Osteoblast-specific overexpression of C5aR1 did not cause abone
phenotype
To investigate whether osteoblast-specific overexpression of C5aR1 influenced osteoblast
activity and bone remodeling, we performed in vitro and in vivo analyses. Western blot analysis
confirmed considerably increased C5aR1 expression in primary osteoblasts derived from
Col1a1-C5aR1 mice after inducing osteogenic differentiation (Fig 1A). Osteoblasts from WT
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 6 / 17
Fig 1. In vitro analysis of WT and Col1a1-C5aR1 osteoblasts. (A) Western blot showing C5aR1 expression in primary osteoblasts of wildtype (WT) and
Col1a1-C5aR1 mice after cultivating for 14 din normal proliferation medium (NM) and osteogenic differentiation medium (ODM). (B) Cell proliferationof WT
and Col1a1-C5aR1 osteoblastsdeterminedby BrdU incorporation. (C) Osteogenic differentiation capacity ofosteoblasts assessed by alkaline-phosphatase
staining after cultivating for 14 din ODM with or without C5a and (D) by the expression of the osteogenic marker genes Alpl (alkaline phosphatase), Ibsp
(bone sialoprotein) and Bglap (bone gamma carboxyglutamate protein/osteocalcin). (E) Western blot showing increased expression of phospho (p)-ERK 1/2
and phospho (p)-p38 after osteoblast stimulation with C5a for 6h. F) Relative gene expression of interleukin (IL)-6, receptor activator of NF-κBligand
(RANKL) and osteoprotegerin (OPG).
*
p<0.05;
***
p<0.001, n = 5–6 per group and treatment.
https://doi.org/10.1371/journal.pone.0179512.g001
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 7 / 17
and Col1a1-C5aR1 mice did not display any difference in proliferation (Fig 1B)or differentia-
tion capacity as demonstrated by alkaline phosphatase staining (Fig 1C)and the expression of
osteogenic differentiation markers (Fig 1D), also in the presence of C5a. Stimulation of WT and
Col1a1-C5aR1 osteoblasts with C5a increased phosphorylation of extracellular signal-regulated
kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) (Fig 1E). In Col1a1--
C5aR1 osteoblasts, phosphorylation of p38 was increased compared to WT osteoblasts, indicat-
ing that the receptor was biologically active. This was confirmed by significantly increased IL-6
and RANKL expression after stimulation of Col1a1-C5aR1 cells with C5a (Fig 1F).
Cross inspection and X-ray examination of the skeleton did not reveal any difference
between 12-week-old and 55-week-old WT and Col1a1-C5aR1 mice (data not shown). μCT
and histomorphometric analyses of the femurs demonstrated that C5aR1 overexpression on
osteoblasts did not influence the bone mass, structural or cellular bone parameters or the bone
formation rate neither in the cortical nor trabecular compartments, indicating that bone devel-
opment and remodeling were not significantly affected (Table 2).
Osteoblast-specific overexpression of C5aR1 did not affect inflammation
in response to fracture
To investigate whether osteoblasts modulate the early inflammation via C5aR1 at the fracture
site, we analyzed the key inflammatory mediator IL-6 and the presence of immune cells locally
in the fracture hematoma. C5a was locally and systemically generated after fracture in WT and
Col1a1-C5aR1 mice without significant differences between the mouse strains (Table 3). In
the isolated fracture model, IL-6 concentration (Table 3)and the numbers of neutrophils
(Ly6G
+
cells), macrophages (F4/80
+
cells) and T-lymphocytes (CD8
+
cells) (Fig 2)were not
significantly altered in Col1a1-C5aR1 mice compared to WT mice.
Because C5a has been reported to trigger posttraumatic systemic inflammation after severe
injury [23], we combined the fracture with an additional thoracic trauma. The combined
trauma significantly enhanced serum IL-6 concentrations in WT and Col1a1-C5aR1 mice,
indicating systemic inflammation (Table 3). In the BAL fluid, C5a and IL-6 were significantly
enhanced in both genotypes (Table 3). The additional thoracic trauma did not influence the
concentration of inflammatory mediators in the fracture hematoma (Table 3)but, according
Table 2. Bone phenotype of WT and Col1a1-C5aR1 mice aged 12 and 55 weeks.
Parameter WT
12 weeks
Col1a1-C5aR1
12 weeks
WT
55 weeks
Col1a1-C5aR1
55 weeks
cortex TMD (g/mm
3
)1282 (±14) 1239 (±7) 1446 (±14) 1409 (±48)
C.Th (mm) 0.20 (±0.01) 0.24 (±0.02) 0.21 (±0.02) 0.21 (±0.03)
trabecular bone BMD (g/mm
3
)200 (±27) 257 (±49) 193 (±12) 179 (±46)
Tb.N (1/mm) 2.8 (±0.5) 3.4 (±0.8) 1.7 (±0.3) 1.8 (±0.3)
Tb.Th (mm) 0.06 (±0.00) 0.06 (±0.01) 0.06 (±0.01) 0.06 (±0.01)
Tb.Sp (mm) 0.19 (±0.02) 0.18 (±0.03) 0.24 (±0.02) 0.25 (±0.01)
Nb.Ob/B.Pm (1/mm) 18.9 (±3.2) 21.5 (±2.6) 15.6 (±6.2) 11.0 (±3.3)
#
Nb.Oc/B.Pm (1/mm) 3.7 (±1.7) 3.1 (±1.9) 2.3 (±0.6) 2.3 (±0.7)
BFR/BS (μm
3
/μm
2
/t) 0.5 (±0.04) 0.7 (±0.16) 0.2 (±0.22) 0.1 (±0.12)
#
WT: wildtype, TMD: tissue mineral density, C.Th: cortical thickness, BMD: bone mineral density, Tb.N: trabecular number, Tb.Th: trabecular thickness, Tb.
Sp: trabecular separation, Nb.Ob/B.Pm: number of osteoblasts per bone perimeter, Nb.Oc/B.Pm: number of osteoclasts per bone perimeter, BFR/BS: bone
formation rate per bone surface. Data are shown as mean ±standard deviation. No data were found to be significantly different (p <0.05) between strains.
#
p<0.05 vs. 12-week-old Col1a1-C5aR1 mice, n = 5–8 per group.
https://doi.org/10.1371/journal.pone.0179512.t002
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 8 / 17
Fig 2. Numberof inflammatory cells in the fracture callus of WT and Col1a1-C5aR mice.(A) Quantification of neutrophils (Ly-6G
+
)at 1d, (B)
representative immunohistological images of neutrophils 1 d after surgery and (C) quantification of neutrophils (Ly-6G
+
) 3 d after surgery. (D) Quantification
of macrophages (F4/80
+
) 3 d and E) T-lymphocytes (CD8
+
) 1 d after surgery of wildtype (WT) and Col1a1-C5aR1 mice. Fx: mice with isolated fracture, Fx
+TXT: mice with combined fracture and thoracic trauma (TXT). Arrows indicate some of the positively Ly-6G
+
stained cells, C: cortex, scale bar 100 μm.
*
p<0.05, n = 6 per group.
https://doi.org/10.1371/journal.pone.0179512.g002
Table 3. C5a and IL-6 concentrations in blood, BAL fluid and fracture hematoma 3 h after surgery.
untreated mice Fracture fracture + TXT
WT Col1a1-C5aR1 WT Col1a1-C5aR1
blood C5a (ng/ml) 0.8 (±0.6) 5.7 (±1.0) 5.3 (±1.5) 6.5 (±1.1) 6.3 (±1.6)
IL-6 (pg/ml) 8.9 (±3.0) 42.8 (±20.3) 57.1 (±28.6) 154.2 (±59.0)*188.2 (±38.4)*
BAL fluid C5a (ng/ml) 8.6 (±1.2) 9.0 (±3.0) 8.6 (±1.4) 13.9 (±2.1)*13.6 (±2.0)*
IL-6 (pg/ml) 3.5 (±0.6) 3.2 (±3.9) 3.1 (±2.8) 123.7 (±55.6)*218.7 (±161.0)*
fracture hematoma C5a (ng/mg protein) - 1.4 (±0.6) 1.2 (±0.6) 1.6 (±0.7) 2.0 (±1.0)
IL-6 (pg/mg protein) - 81.9 (±49.2) 80.4 (±51.8) 53.2 (±27.0) 35.3 (±26.4)
BAL: bronchoalveolar lavage, WT, wildtype, TXT: thorax trauma. Data are shown as mean ±standard deviation. C5a was analyzed in plasma and IL-6 was
analyzed in serum.
*
p<0.05 vs. group without TXT, n = 5–6 per group.
https://doi.org/10.1371/journal.pone.0179512.t003
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 9 / 17
to our previous studies [14,15,19,24], increased the recruitment of neutrophils to the fracture
site in WT mice (Fig 2). However, the osteoblast-specific overexpression of C5aR1 did not
affect inflammatory parameters in the combined trauma model (Table 3,Fig 2).
Osteoblast-specific overexpression of C5aR1 significantly impaired
bone formation in the fracture callus
To investigate callus formation, we analyzed the tissue composition in the repair phase on day
14, when callus tissue is abundantly formed by intramembranous and endochondral ossifica-
tion. In the isolated fracture model, we did not find significant differences in the content of
cartilage and bone between the genotypes (Fig 3A–3D). Notably, the number of osteoblasts
was significantly decreased whereas the number of osteoclasts increased in the callus of mice
with an osteoblast-specific C5aR1 overexpression (Fig 3E–3G). The additional thoracic trauma
Fig 3. Fracturehealing in WT and Col1a1-C5aR1 mice 14 dafter surgery. (A) Representative histological images of wildtype (WT) and Col1a1-C5aR1
mice with isolated fracture and fracture with additional thoracic trauma (TXT). (B) Callus area, (C) amount of osseous tissue and (D) amount of cartilage. (E)
Number of osteoblasts per bone perimeter(N.Ob/B.Pm)and (F) number of osteoclasts per bone perimeter (N.Oc/B.Pm) of WT and Col1a1-C5aR1 mice. (G)
Representative tartrate-resistant acid phosphatase (TRAP) staining of fractured calli of WT and Col1a1-C5aR1 mice with isolated fracture. Fx: mice with
isolated fracture, Fx+TXT: mice with combined fracture and thoracic trauma. C: cortex, scale bar 100 μm,
*
p<0.05, n=6per group.
https://doi.org/10.1371/journal.pone.0179512.g003
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 10 /17
slightly reduced the amount of bone and decreased the number of osteoblasts in the callus
without significant differences between both genotypes (Fig 3C and 3E). Immunostaining
revealed intense staining for Runx2 and osteocalcin of osteoblasts on newly formed bone tra-
beculae in all groups without significant differences between genotypes, indicating undis-
turbed osteogenic differentiation (Fig 4). We did not detect significant differences in the
expression of RANKL, but found less OPG-positive osteoblasts in mice with Col1a1-C5aR1
overexpression.
In WT mice with isolated fracture, most of the cartilage was transformed to bone after 21
days. However, in Col1a1-C5aR1 mice, bone content, BMD and the flexural rigidity were sig-
nificantly reduced (Fig 5). We found more osteoclasts in the callus of Col1a1-C5aR1 mice,
whereas osteoblast numbers were not different compared to WT mice (Fig 5G–5I). In Col1a1--
C5aR1 mice, the additional thoracic trauma induced significantly more pronounced effects
compared to WT mice as demonstrated by significantly reduced bone content and flexural
rigidity (Fig 5C and 5F).
Fig 4. Representative immunohistological images of bone markersin the fracture callus of WT and Col1a1-C5aR1mice 14 dafter surgery. (A)
Runx2,(B) osteocalcin, (C) receptor activator of nuclear factor-κBligand (RANKL) and (D) osteoprotegerin (OPG). WT: wildtype, Fx: mice with isolated
fracture,Fx+TXT: mice with combined fracture and thoracic trauma (TXT). Arrows indicate some of the positively stained cells, C: cortex, scale bar 100 μm.
https://doi.org/10.1371/journal.pone.0179512.g004
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 11 /17
After 25 days, fractured bones were completely healed in both genotypes with isolated
fracture (Fig 6). However, at this time point, the strong effect of the thoracic trauma in
Fig 5. Fracturehealing in WT and Col1a1-C5aR1 mice 21 dafter surgery. (A) Representative histological images of wildtype (WT) and Col1a1-C5aR1
mice with isolated fracture and fracture with additional thoracic trauma (TXT). (B) Callus area, (C) amount of osseous tissue and (D) amount of cartilage. (E)
Bone mineral density (BMD). (F) Relative flexural rigidity. (G) Number of osteoblasts per bone perimeter(N.Ob/B.Pm) and (H) number of osteoclastsper
bone perimeter (N.Oc/B.Pm) of WT and Col1a1-C5aRmice. (I) Representative tartrate-resistant acid phosphatase (TRAP) staining of fracturedcalli of WT
and Col1a1-C5aR1 mice with isolated fracture.Fx: mice with isolated fracture, Fx+TXT: mice with combined fracture and thoracic trauma. C: cortex, scale
bar 100 μm,
*
p<0.05;
**
p<0.005;
***
p<0.001, n = 6–7 per group.
https://doi.org/10.1371/journal.pone.0179512.g005
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 12 /17
Col1a1-C5aR1 mice was still clear as demonstrated by significantly reduced flexural rigidity of
the fractured femurs, increased callus area and atendential increase in the cartilaginous tissue
area (Fig 6).
Discussion
The objective of the present study was to unravel the role of the C5aR1 in osteoblasts regarding
inflammation and bone formation post fracture using mice with an osteoblast-specific overex-
pression of C5aR1. Transgenic Col1a1-C5aR1 mice displayed normal bone development and
turnover. However, fracture healing both in amodel of uncomplicated fracture and in amodel
after severe additional trauma was considerably impaired in transgenic mice, indicating that
C5aR1 signaling in osteoblasts may be crucially involved in bone formation under pathological
conditions.
We first characterized the bone phenotype of Col1a1-C5aR1 mice to investigate whether
C5aR1 overexpression affects bone development and turnover. The morphology of the skele-
ton and the density and structural parameters of cortical and trabecular bone were unaffected
in both young and elderly Col1a1-C5aR1 mice. Col1a1-C5aR1 osteoblasts derived from these
mice exhibited undisturbed proliferation and differentiation capacities, including after stimu-
lation of the receptor with its ligand C5a. These results demonstrated that osteoblast-specific
C5aR1 overexpression did not alter bone development or turnover. However, this did not
exclude an important role for the C5aR1 under physiological conditions. It was demonstrated
that osteoblasts produce the key complement protein C5 also in the absence of inflammatory
Fig 6. Fracture healing in WT and Col1a1-C5aR1 mice 25 dafter surgery. (A) Callus area, (B) amount of osseous
tissue, (C) amount of cartilageand (D) relative flexural rigidity of wildtype (WT) and Col1a1-C5aR1 mice. Fx: mice with
isolated fracture, Fx+TXT: mice with combined fracture and thoracic trauma (TXT).
*
p<0.05, n = 6 per group.
https://doi.org/10.1371/journal.pone.0179512.g006
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 13 /17
stimuli and that osteoclasts can effectively cleave C5 to biologically active C5a [12]. Confirm-
ing this, Andrades et al. found C5 together with other complement components expressed in
the bone growth plate and suggested that complement might contribute to the transformation
of cartilage to bone during endochondral ossification [25]. Indeed, previous examination of
the bone phenotype of C5-deficient mice demonstrated that the absence of C5 had an impact
on cortical bone [2]. Twelve-week-old C5-deficient mice exhibited decreased bending stiffness
and increased growth plates, confirming that C5a signaling could play aregulatory role in
bone development, however, further studies are necessary to dissect the molecular mecha-
nisms [2].
Confirming previous data that C5aR1 signaling induces apro-inflammatory and osteoclas-
togenic response in osteoblasts [10,12], our results revealed that IL-6 and RANKL expression
were significantly increased in Col1a1-C5aR1 osteoblasts in response to C5a. In agreement
with findings in immune cells [26–28]and MSCs [11], we showed that the C5aR1 downstream
signaling in osteoblasts involved ERK1/2 and p38 phosphorylation, which is known to regulate
the expression of inflammatory cytokines, including IL-6 [27,29]. These data suggest that the
C5aR1 axis in osteoblasts may be particularly important under inflammatory conditions. In
accordance with this, it was shown that C5aR1-knockout mice were protected against ost-
eoarthritis [30]and that bone loss in bacteria-induced periodontitis was associated with an
increased C5aR1 activity in osteoblasts [31]. We previously demonstrated that osteoblasts
strongly upregulated C5aR1 during the early phase after bone fracture [1]. Therefore, we
hypothesized, that C5a, which is known to be abundantly generated locally and systemically in
response to tissue injury [32–34], may induce an immune-modulatory response in osteoblasts
during fracture healing. Our results revealed that the concentration of IL-6, akey cytokine in
the local immune response after bone fracture and in posttraumatic systemic inflammation
[35–38], was neither locally nor systemically altered in Col1a1-C5aR1 mice. Additionally, the
recruitment of innate and adaptive immune cells to the fracture site was not significantly influ-
enced, suggesting that C5aR1 signaling in osteoblasts did not modulate the early immune
response after fracture. Apossible reason may be the low number of osteoblasts compared to
the high number of active immune cells during this early stage. However, bone healing was
considerably disturbed in the later healing stages in Col1a1-C5aR1 mice. The bone content
and mineral density and the mechanical properties of the fracture callus were significantly
reduced. The reduced bone fraction may be primarily caused by asignificantly increased num-
ber of osteoclasts observed after 14 and 21 days in the fracture callus of Col1a1-C5aR1 mice.
These observations are in agreement with the present and previous in vitro data, demonstrat-
ing that C5a significantly enhanced the RANKL/OPG ratio in osteoblasts, thereby promoting
osteoclastogenesis [12]. Immunohistochemistry did not reveal obvious differences in RANKL
expression, but OPG expression was clearly reduced in the callus of Col1a1-C5aR1 mice.
These results are confirmed by other authors demonstrating that C5aR1 is important for osteo-
clastogenesis [1,10,12]. Bone marrow cells from C5aR1-deficient mice and abrogating C5aR1
signaling in WT cells with aC5aR1 antibody resulted in decreased osteoclastogenesis, whereas
stimulation of WT or C3-deficent bone marrow cells with C5a enhanced osteoclastogenesis
[13]. Moreover, IL-6 is probably involved in the differentiation of osteoclasts regulated by
complement, because IL-6 expression was decreased in bone marrow cells from C3aR/C5aR1-
deficent mice [13]. In Col1a1-C5aR1 mice, osteoblast numbers in the fracture callus were
reduced on day 14 but were unchanged on day 21 and the expression of the osteoblastic differ-
entiation markers Runx2 and osteocalcin was unaltered, suggesting that osteoblast activity
might not be considerably affected, which was confirmed by our in vitro data. However, this
needs further investigation. Moreover, to the best of our knowledge, no literature data exist
about the role of the second C5a receptor, C5aR2, in bone metabolism and fracture healing.
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 14 /17
Concluding, the data suggest that C5aR1 signaling in osteoblasts may crucially modulate oste-
oclast formation and activity and thereby increase bone resorption during fracture healing by
inducing the expression of osteoclastic factors, including RANKL and IL-6.
We also investigated healing in acombined model of fracture and thoracic trauma, which
induces aposttraumatic systemic inflammatory response, resulting in impaired fracture heal-
ing [14,15,19,24]. Previously, we demonstrated that blockade of the C5aR1 by aspecific antag-
onist during the early inflammatory phase abolished the negative effects of the severe trauma
on fracture healing, indicating that C5a abundantly generated during posttraumatic inflamma-
tion may crucially contribute to trauma-induced disturbed fracture healing [39]. The com-
bined fracture and thoracic trauma induced asystemic posttraumatic inflammatory response
and impaired bone healing in both WT and Col1a1-C5aR1 mice, as demonstrated by increased
IL-6 serum levels and by significantly reduced bone content and mechanical properties of the
fracture callus, respectively, in both mouse strains. The impairment of fracture healing by the
additional thoracic trauma was more pronounced in mice with the osteoblast-specific overex-
pression of C5aR1, confirming the significance of the C5a/C5aR1 axis in osteoblasts during
fracture healing. After 25 days, when the fractures were completely healed in WT mice,
Col1a1-C5aR1 mice still exhibited asignificantly reduced bending stiffness, suggesting adetri-
mental impact of C5aR1 signaling in trauma-induced impaired bone regeneration.
In conclusion, the present data together with our previous experiments [1,12,15,24,39,40]
suggest acrucial role of the C5aR1 in fracture healing and implies that C5aR1 inhibition
may be asuitable strategy to prevent fracture-healing complications particularly after severe
trauma.
Acknowledgments
The authors acknowledge the excellent technical assistance of Sonja Braunmu¨ller, Bettina
Stahl, Sevil Essig, Marion Tomo, Ursula Maile, Stefanie Riesemann and Helga Bach.
Author Contributions
Conceptualization: MHL AI.
Data curation: AI.
Formal analysis: SB AK YH.
Funding acquisition: TS MA MHL AI.
Investigation: SB AK YH AL TS.
Methodology: AL TS MA MHL AI.
Project administration: SB AK AI.
Resources: TS MA MHL AI.
Supervision: AK MHL AI.
Validation: SB AK YH AL AI.
Visualization: SB AK YH.
Writing –original draft: SB AK YH AI.
Writing –review &editing: SB AK YH AL TS MA MHL AI.
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 15 /17
References
1. Ignatius A, EhrnthallerC, Brenner RE, Kreja L, Schoengraf P, Lisson P, et al. The anaphylatoxin recep-
tor C5aR is present during fracture healing in rats and mediates osteoblast migration in vitro. JTrauma.
2011 Oct; 71(4):952–60. PMID: 21460748.https://doi.org/10.1097/TA.0b013e3181f8aa2d
2. EhrnthallerC, Huber-Lang M, Nilsson P, Bindl R, Redeker S, Recknagel S, et al. Complement C3 and
C5 deficiency affects fracture healing. PLoS One. 2013 Nov 18; 8(11):e81341. https://doi.org/10.1371/
journal.pone.0081341 PMID: 24260573.
3. Scho
¨ngraf P, Lambris JD, Recknagel S, Kreja L, Liedert A, Brenner RE, et al. Does complement play a
role in bone development and regeneration? Immunobiology. 2013 Jan; 218(1):1–9.PMID: 22464814.
https://doi.org/10.1016/j.imbio.2012.01.020
4. Ricklin D, Hajishengallis G, Yang K, Lambris JD. Complement: a key system for immune surveillance and
homeostasis. Nat Immunol. 2010 Sep; 11(9):785–97.PMID: 20720586.https://doi.org/10.1038/ni.1923
5. Amara U, RittirschD, Flierl M, Bruckner U, Klos A, Gebhard F, et al. Interactionbetween the coagulation
and complement system. Adv Exp Med Biol. 2008; 632: 71–9. PMID: 19025115.
6. Klos A, Tenner AJ, Johswich KO, Ager RR, Reis ES, Ko
¨hl J. The role of the anaphylatoxins in health
and disease. Mol Immunol. 2009; Sep; 46(14):2753–66. PMID: 19477527.https://doi.org/10.1016/j.
molimm.2009.04.027
7. Manthey HD, Woodruff TM, Taylor SM, Monk PN. Complement component 5a (C5a). Int JBiochem
Cell Biol. 2009 Nov; 41(11):2114–7. PMID: 19464229.https://doi.org/10.1016/j.biocel.2009.04.005
8. Monk PN, Scola AM, Madala P, Fairlie DP. Function, structure and therapeutic potential of complement
C5a receptors. Br JPharmacol. 2007 Oct; 152(4):429–48. PMID: 17603557.https://doi.org/10.1038/sj.
bjp.0707332
9. Gerard NP, Hodges MK, Drazen JM, Weller PF, Gerard C. Characterization of areceptor for C5a ana-
phylatoxin on human eosinophils. JBiol Chem. 1989 Jan 25; 264(3):1760–6. PMID: 2912983.
10. Pobanz JM, Reinhardt RA, Koka S, Sanderson SD. C5a modulation of interleukin-1 beta-induced inter-
leukin-6 production by human osteoblast-like cells. JPeriodontalRes. 2000 Jun; 35(3):137–45. PMID:
10929868.
11. Schraufstatter IU, Discipio RG, Zhao M, Khaldoyanidi SK. C3a and C5a are chemotactic factors for
human MSCs, which cause prolonged ERK1/2 phosphorylation. JImmunol. 2009 Mar 15; 182
(6):3827–36. PMID: 19265162.https://doi.org/10.4049/jimmunol.0803055
12. Ignatius A, Schoengraf P, Kreja L, Liedert A, Recknagel S, Kandert S, et al. Complement C3a and C5a
modulate osteoclast formation and inflammatoryresponse of osteoblastsin synergism with IL-1β. J Cell
Biochem. 2011 Sep; 112(9):2594–605. PMID: 21598302.https://doi.org/10.1002/jcb.23186
13. Tu Z, Bu H, Dennis JE, Lin F. Efficient osteoclast differentiation requires local complement activation.
Blood. 2010 Nov 25; 116(22):4456–63. PMID: 20709903 https://doi.org/10.1182/blood-2010-01-263590
14. Kemmler J, Bindl R, McCook O, Wagner F, Gro
¨ger M, Wagner K, et al. Exposure to 100% Oxygen abol-
ishes the impairment of fracture healing after thoracic trauma. PLoS One. 2015Jul 6; 10(7):e0131194.
https://doi.org/10.1371/journal.pone.0131194 PMID: 26147725.
15. Recknagel S, Bindl R, Brochhausen C, Go
¨ckelmann M, Wehner T, Schoengraf P, et al. Systemic
inflammationinduced by athoracic trauma alters the cellular composition of the early fracture callus. J
Trauma Acute Care Surg. 2013 Feb; 74(2):531–7. PMID: 23354247.https://doi.org/10.1097/TA.
0b013e318278956d
16. Ramakers C, Ruijter JM, Deprez RH, Moorman AF. Assumption-free analysis of quantitativereal-time
polymerase chain reaction (PCR) data. Neurosci Lett. 2003 Mar 13; 339(1):62–6. PMID: 12618301.
17. Ro
¨ntgen V, Blakytny R, Matthys R, Landauer M, Wehner T, Go
¨ckelmann M, et al. Fracture healing in
mice under controlled rigid and flexible conditionsusing an adjustable external fixator. JOrthop Res.
2010 Nov; 28(11):1456–62. PMID: 20872581.https://doi.org/10.1002/jor.21148
18. Kno
¨ferl MW, Liener UC, Seitz DH, Perl M, Bru¨ckner UB, Kinzl L, et al. Cardiopulmonary, histological,
and inflammatoryalterations after lung contusion in anovel mouse model of blunt chest trauma. Shock.
2003 Jun; 19(6):519–25. PMID: 12785006.https://doi.org/10.1097/01.shk.0000070739.34700.f6
19. Kovtun A, Bergdolt S, Wiegner R, Radermacher P, Huber-Lang M, Ignatius A. The crucial role of neutro-
phil granulocytes in bone fracture healing. Eur Cell Mater. 2016 Jul 25; 32:152–62. PMID: 27452963.
20. Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Mu¨ller R. Guidelines for assess-
ment of bone microstructure in rodents using micro-computed tomography. JBone Miner Res. 2010
Jul; 25(7):1468–86. PMID: 20533309.https://doi.org/10.1002/jbmr.141
21. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Bone histomorphometry:
Standardization of nomenclature, symbols and units. JBone Miner Res. 1987 Dec; 2(6):595–610.
PMID: 3455637.https://doi.org/10.1002/jbmr.5650020617
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
PLOS ONE |https://doi.org/10.1371/journal.pone.0179512 June 14, 2017 16 /17
22. Morgan EF, Mason ZD, Chien KB, Pfeiffer AJ, Barnes GL, Einhorn TA, et al. Micro-computed tomogra-
phy assessment of fracture healing: Relationships among callus structure, composition, and mechani-
cal function. Bone. 2009 Feb; 44(2):335–44. PMID: 19013264.https://doi.org/10.1016/j.bone.2008.10.
039
23. Huber-Lang M, Gebhard F, Schmidt CQ, Palmer A, Denk S, Wiegner R. Complement therapeutic strat-
egies in trauma, hemorrhagic shock and systemic inflammation—closingPandora’s box? Semin Immu-
nol. 2016 Jun; 28(3):278–84. PMID: 27157701.https://doi.org/10.1016/j.smim.2016.04.005
24. Recknagel S, Bindl R, Kurz J, Wehner T, EhrnthallerC, Knoferl MW, et al. Experimental blunt chest
trauma impairs fracture healing in rats. JOrthop Res. 2011 May; 29(5):734–9. PMID: 21437953.https://
doi.org/10.1002/jor.21299
25. Andrades JA1, Nimni ME, Becerra J, Eisenstein R, Davis M, Sorgente N. Complement proteins are
present in developing endochondral bone and may mediate cartilagecell death and vascularization.
Exp Cell Res. 1996 Sep 15; 227(2):208–13. PMID: 8831558 https://doi.org/10.1006/excr.1996.0269
26. Chiou WF, Tsai HR, Yang LM, Tsai WJ. C5a differentially stimulates the ERK1/2 and p38 MAPK phos-
phorylation throughindependent signaling pathwaysto induced chemotactic migration in RAW264.7
macrophages. Int Immunopharmacol. 2004 Oct; 4(10–11):1329–41. PMID: 15313431.https://doi.org/
10.1016/j.intimp.2004.05.017
27. Riedemann NC, Guo RF, Hollmann TJ, Gao H, Neff TA, Reuben JS, et al. Regulatoryrole of C5a in
LPS-induced IL-6 production by neutrophils during sepsis.FASEB J. 2004 Feb; 18(2):370–2. PMID:
14688199.https://doi.org/10.1096/fj.03-0708fje
28. RousseauS, Dolado I, Beardmore V, Shpiro N, MarquezR, Nebreda AR, et al. CXCL12 and C5a trigger
cell migrationvia aPAK1/2-p38alpha MAPK-MAPKAP-K2-HSP27 pathway.Cell Signal. 2006 Nov; 18
(11):1897–905. PMID: 16574378.https://doi.org/10.1016/j.cellsig.2006.02.006
29. Tuyt LM, Dokter WH, BirkenkampK, Koopmans SB, Lummen C, Kruijer W, et al. Extracellular-regulated
kinase 1/2, Jun N-terminal kinase, and c-Jun are involved in NF-kappa B-dependent IL-6 expression in
human monocytes. J Immunol. 1999 Apr 15; 162(8):4893–902. PMID: 10202034.
30. Banda NK, Hyatt S, Antonioli AH, White JT, Glogowska M, Takahashi K, et al. Role of C3a receptors,
C5a receptors, and complement protein C6 deficiency in collagen antibody-induced arthritis in mice. J
Immunol. 2012 Feb 1; 188(3):1469–78. PMID: 22205026.https://doi.org/10.4049/jimmunol.1102310
31. Liang S, Krauss JK, Domon H, McIntosh ML, Hosur KB, Qu H, et al. The C5a receptor impairs IL-12–
dependent clearance of Porphyromonas gingivalis and is required for induction of periodontal bone
loss. JImmunol. 2011 Jan 15; 186(2):869–77. PMID: 21149611.https://doi.org/10.4049/jimmunol.
1003252
32. Dong Q, Sun L, Peng L, Yan B, Lv J, Wang G, et al. Expressionof C5a and its receptor following spinal
cord ischemia reperfusion injury in the rat. Spinal Cord. 2015 Aug; 53(8):581–4. PMID: 25917954.
https://doi.org/10.1038/sc.2015.65
33. Kanse SM, Gallenmueller A, Zeerleder S, Stephan F, Rannou O, Denk S, et al. Factor VII-activating
protease is activated in multiple trauma patients and generates anaphylatoxin C5a. JImmunol. 2012
Mar 15; 188(6):2858–65. PMID: 22308306.https://doi.org/10.4049/jimmunol.1103029
34. LeinhaseI, Holers VM, Thurman JM, Harhausen D, Schmidt OI, Pietzcker M, et al. Reduced neuronal
cell death after experimental brain injury in mice lacking afunctional alternative pathway of complement
activation. BMC Neurosci. 2006 Jul 14; 7:55. PMID: 16842619.https://doi.org/10.1186/1471-2202-7-55
35. Gebhard F, Pfetsch H, Steinbach G, Strecker W, Kinzl L, Bruckner UB. Is interleukin 6an early marker
of injury severity following major trauma in humans? Archives of surgery. 2000 Mar; 135(3):291–5.
PMID: 10722030.
36. Kolar P, Schmidt-Bleek K, Schell H, Gaber T, Toben D, SchmidmaierG, et al. The early fracture hema-
toma and its potentialrole in fracture healing. Tissue Eng Part BRev. 2010 Aug; 16(4):427–34. PMID:
20196645.https://doi.org/10.1089/ten.TEB.2009.0687
37. Marsell R, Einhorn TA. The biology of fracture healing. Injury. 2011 Jun; 42(6):551–5. PMID: 21489527.
https://doi.org/10.1016/j.injury.2011.03.031
38. Yang X, Ricciardi BF, Hernandez-Soria A, Shi Y, Pleshko Camacho N, Bostrom MP. Callus mineraliza-
tion and maturationare delayed during fracture healing in interleukin-6 knockout mice. Bone. 2007 Dec;
41(6):928–36. PMID: 17921078.https://doi.org/10.1016/j.bone.2007.07.022
39. Recknagel S, Bindl R, Kurz J, Wehner T, Schoengraf P, EhrnthallerC, et al. C5aR-antagonist signifi-
cantly reduces the deleterious effect of ablunt chest trauma on fracture healing. JOrthop Res. 2012
Apr; 30(4):581–6. PMID: 21922535.https://doi.org/10.1002/jor.21561
40. EhrnthallerC, Ignatius A, Gebhard F, Huber-Lang M. New insights of an old defense system: structure,
function, and clinical relevance of the complement system. Mol Med. 2011 Mar-Apr; 17(3–4):317–29.
PMID: 21046060.https://doi.org/10.2119/molmed.2010.00149
Osteoblast-specific overexpression of C5aR1 impairs fracture healing
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