Received: 6 September 2020 | Revised: 27 November 2020
DOI: 10.1111/odi.13745
ORIGINAL ARTICLE
HIF-1α regulates osteoclast activation and mediates
osteogenesis during mandibular bone repair via CT-1
Yuanye Tian1,2 | Qi Shao1,2 | Yi Tang1,2 | Xinzhao Li1,2 | Xin Qi1,2 |
Runyang Jiang1,2 | Yi Liang1,2 | Feiwu Kang1,2
1
Department of Oral and Maxillofacial
Surgery, School & Hospital of Stomatology,
Tongji University, Shanghai, China
2
Shanghai Engineering Research Center
of Tooth Restoration and Regeneration,
Shanghai, China
Correspondence
Feiwu Kang, Department of Oral and
Maxillofacial Surgery, School & Hospital of
Stomatology, Tongji University, Shanghai
Engineering Research Center of Tooth
Restoration and Regeneration, 399 Middle
Yanchang Road, Shanghai, 200072, China.
Email: kfw@tongji.edu.cn
Funding information
National Natural Science Foundation of
China, Grant/Award Number: 81670961
1 | I NTRO D U C TI O N
The treatment of bone malocclusion often requires a combination
of orthodontics and orthognathic surgery. This surgery mainly in-
volves several types of osteotomies to move the jaw block in order
Tian and Shao contributed equally as first authors.
428 | © 2020 Wiley Periodicals LLC
| Accepted: 30 November 2020
Abstract
Objectives: Hypoxia is one of the characteristics of microenvironmental changes
after orthognathic surgery for fractures. HIF-1α is a main regulator of the hypoxic
response and plays a crucial role in bone formation, remodelling, and homeostasis.
Osteoclasts participate in bone absorption and affect osteogenesis, and osteoclasts
differentiate in a path from the oxygen-rich bone marrow to oxygen-deficient bone
lesions. Thus, we aimed to study the key functions of HIF-1α in osteoclasts during
mandibular healing after osteotomy.
Materials and Methods: The function of HIF-1α in osteoclasts during fracture healing
in osteoclast-specific HIF-1α-conditional-knockout mice was investigated in mandib-
ular osteotomy. Primary osteoclasts were used to explore the expression of HIF-1α
and cardiotrophin-1 (CT-1) at both the mRNA and protein levels. The ability of BMSCs
co-cultured with conditioned media from osteoclast-specific HIF-1α-knockout pri-
mary osteoclasts was detected using osteoclast-mediated osteogenesis experiments.
Results: Hypoxia-inducible factor-1α increased osteoclastogenesis and bone resorp-
tion, and a delay in bone healing was found in osteoclast-specific HIF-1α-conditional-
knockout mice compared with normal mice. HIF-1α-knockout primary osteoclasts
inhibited bone resorption and CT-1 expression, and HIF-1α enhanced the osteoclast-
mediated stimulation of BMSC differentiation by secreting CT-1.
Conclusions: Hypoxia-inducible factor-1α can play a key role in the physiology and
pathogenesis of bone resorption by promoting osteoclastogenesis during fracture
and influencing osteogenesis through CT-1 during bone healing.
KEYWORDS
coupling factor, crosstalk, hypoxia, mandible, osteoclastogenesis, osteogenesis
to correct a wide spectrum of dentofacial deformities (Patel &
Novia, 2007). These osteotomies inevitably create a wedge-shaped
osteotomy gap. Thus, bone gap repair is an important process after
surgery. All patients of clinical orthodontic and orthognathic doctors
expect an accelerated and well-done stable repair.
Bone repair is a multistage and complicated process (Ghiasi
et al., 2017) involving osteoclastic bone resorption and osteoblastic
wileyonlinelibrary.com/journal/odi Oral Diseases. 2022;28:428–441.
TIAN et al.
bone formation (Runyan & Gabrick, 2017). The mandible is formed via
intramembranous bone formation directly from mesenchymal conden-
sations without a cartilaginous template (Scott & Hightower, 1991),
and its morphogenesis depends on the crosstalk of osteoblasts and
osteoclasts. Osteoclasts can synthesize and secrete coupling factors,
including the proteins secreted by osteoclasts, membrane-bound
proteins on the osteoclast surface, and exosome-associated proteins
and miRNAs released by osteoclasts (Sims & Martin, 2019; Sims &
Vrahnas, 2014), but the coupling factors that influence mandible repair
are uncertain. Orthognathic surgery might lead to haematoma forma-
tion, hypoxic tension and local hypoxia. Therefore, hypoxia-inducible
factor-1α (HIF-1α) expression might be enhanced at the fracture site. In
recent years, many studies have focused on the role of HIF-1α in frac-
ture repair (Huang et al., 2015; Maes et al., 2010; Miclau et al., 2017),
and in particular, on the role of HIF-1α in osteoblasts or osteoclasts
(Knowles, 2015, 2017). Our previous research showed that HIF-1α
mediates osteoclast-induced mandibular condylar growth (Tang
et al., 2020). However, the functions of HIF-1α in osteoclasts during
mandibular repair, particularly how HIF-1α influences the crosstalk be-
tween osteoclasts and osteoblasts, are not well understood.
Cardiotrophin-1 (CT-1) is a gp130-signalling cytokine of the in-
terleukin-6 (IL-6) superfamily (Jin et al., 1996) that is synthesized and
secreted by osteoclasts as a coupling factor (Walker et al., 2008).
CT-1 can influence the function of osteoclasts (Richards et al., 2000)
and stimulate bone formation through multiple mechanisms (Walker
et al., 2010). In our study, we hypothesized that HIF-1α regulates
the function of osteoclasts and osteoclast-mediated osteogenesis
through CT-1. The aim of this study was to elucidate the functional
role of HIF-1α in osteoclasts and the mechanism by which HIF-1α
in osteoclasts regulates mandibular bone repair after osteotomy via
CT-1.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Animals and ethics statement
Mice were purchased from the Jackson Laboratory. Osteoclast-
specific HIF-1α-conditional-knockout (Cko: HIF-1αflox/flox; Ctsk
cre+) mice were generated by intercrossing the mice homozygous
for a floxed HIF-1α allele with mice harbouring Cre in the Cathepsin
K locus (Ctsk cre+). We compared Cko mice with wild-type con-
trols (Ctrl: HIF-1αflox/flox; Ctsk cre-). The animals were handled
according to the National Institutes of Health's Guide to the Care
and Use of Laboratory Animals. The Animal Ethics Committee of the
Stomatological Hospital Affiliated to Tongji University approved all
procedures involving animals.
2.2 | Mandibular osteotomy
One-month-old Cko or Ctrl mice were anaesthetized by inhalation
of 4.0% sevoflurane, and anaesthesia was maintained with 3.0%
| 429
sevoflurane. A 2-cm skin incision was made on the lower edge of
the chin. We carefully dissected the musculature and periosteum,
exposing the bone between the two foramina. A sagittal incision was
made throughout the cortical bone to perform a parasymphyseal
mandibular osteotomy. A 1-mm-diameter drill bit was used to rotate
the dental instrument at high speed to quickly grind out the inci-
sion, and cooling with condensed saline was performed throughout
the procedure. Conventional haemostasis and suturing were sub-
sequently accomplished. At the end of anaesthesia, the mice were
returned to their cages.
2.3 | X-ray and micro-computed tomography
X-ray and computed tomography were used to quantify bone re-
modelling by using a micro-CT system (Micro-CT 50; Scanco
Medical) and related analysis software. We selected the entire re-
gion of the bone defect for analysis, and 40 Z planes were imaged.
Histomorphometric values included the bone volume fraction (BV/
TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp),
mean/density and trabecular number (Tb.N). Six male mice in the
Ctrl or Cko group at 1, 3 and 5 weeks postsurgery were euthanized,
and the mandible was collected for scanning analysis.
2.4 | Histopathological sample preparation
After scanning analysis with a micro-CT system, the mandible was
completely separated, fixed in 4% paraformaldehyde for 48 hr, de-
calcified with 15% (wt/vol) ethylenediaminetetraacetic acid (EDTA),
and then embedded in paraffin with automatic equipment. The par-
affin section was approximately 5 μm thick.
2.5 | Histological staining
Sections were stained with an H&E Staining Kit (KeyGen) and tar-
trate-resistant acid phosphatase (TRAP) staining kit (Sigma-Aldrich).
An alkaline phosphatase (ALP) staining kit (Jiancheng) was utilized to
detect bone formation. Osteoclasts were identified by TRAP stain-
ing, and TRAP-positive osteoclasts were counted using multiple
sections.
2.6 | Immunohistochemistry and
immunofluorescence staining
The primary antibody used was rabbit anti-osteocalcin (OCN, 1:200;
Boster), and the secondary antibody was goat anti-rabbit IgG (1:200;
KeyGen). The slides were visualized with a DAB detection kit and
then stained with haematoxylin. For immunofluorescence stain-
ing, the primary antibodies were rabbit anti-cardiotrophin-1 (1:200;
Affinity), rabbit anti-HIF-1α (1:200; Abcam), and mouse anti-CTSK
430 | TIAN et al.
(1:200; BBI). Then, the slides were incubated with DyLight 488-con-
jugated goat anti-mouse IgG (1:500; Abbkine) or DyLight 594-conju-
gated goat anti-rabbit IgG (1:500; Abbkine) followed by DAPI (1:800;
Sigma-Aldrich).
2.7 | Cell culture, staining and bone resorption assay
Bone marrow monocytes (BMMs) isolated from Cko and Ctrl mouse
femurs and tibias were treated with 100 ng/ml receptor activa-
tor of nuclear factor-κB ligand (RANKL; R&D Systems) and 30 ng/
ml mouse macrophage colony-stimulating factor (M-CSF; R&D
Systems) for 6 days. All serum- or supplement-containing media
were replaced every 2 days. BMMs were treated with 100 μM CoCl2
(Sigma-Aldrich) for the last 48 hr to mimic hypoxic conditions. The
cells were incubated with a TRAP staining kit, toluidine blue stain-
ing kit (Solarbio) and FITC Phalloidin (Sigma-Aldrich). Osteoclasts
were identified by TRAP staining, and TRAP-positive osteoclasts
were counted using multiple sections. For the bone resorption assay,
BMMs were seeded on bone slices in 24-well plates and cultured
with osteoclastogenic medium for 14 days. The medium was aspi-
rated, and sodium hypochlorite was used for bleaching three times.
2.8 | Transient transfection
Bone marrow monocytes were seeded in 6-well plates in α-MEM
for 24 hr. The medium was changed to new α-MEM containing
10% FBS 1 hr before transient transfection. Cells at 70%-80%
confluence were transfected with 50 nM CT-1 Silencer (siCT-1, 5′-
GACCAAATATGCAGAACAA-3′) to knock down CT-1 expression
or negative control (siNC). RiboTM RNA Smart Silencer (RiboBio
Company) and Lipofectamine 3,000 (Invitrogen) were used accord-
ing to the manufacturer's instructions. After transfection for 24 hr,
the medium was aspirated and replaced with conditional medium for
culture.
2.9 | RNA isolation and qPCR
BMMs were seeded into 6-well plates as described before. Mandibles
from the sham group or Ctrl osteotomy group mice obtained at 3,
7 and 14 days postsurgery were ground to homogenate with a ho-
mogenizer (Tissuelyser-24, Jingxin). Then, RNA was isolated from
cultured cells or mandibular tissue with TRIzol Reagent (TaKaRa)
according to the manufacturer's instructions. The RNA sample was
tested with a NanoDrop 2000 spectrometer (NanoDrop Technology)
for purity and concentration. First-strand cDNA was transcribed
from 1,000 ng RNA by PrimeScript RT reagent Kit with gDNA Eraser
(TaKaRa). Quantitative real-time polymerase chain reaction (PCR)
was performed with the Applied Biosystems QuantStudio 6 with
SYBR Premix Ex Taq II kit (TaKaRa) in triplicate. The amplification
profile consisted of 30 s at 95°C, followed by 45 cycles at 95°C for
10 s and 62°C for 35 s. The primers for PCR were purchased from
Sango Biotech. β-Actin was used for normalization, and mRNA ex-
pression was calculated using the 2−ΔΔCT method. The sequences of
the primers used are shown in Appendix Table S1.
2.10 | Microarray analysis
Total RNA was extracted from Cko and Ctrl mouse bone marrow
macrophages induced with RANKL (100 ng/ml) and M-CSF (30 ng/
ml) for 6 days. Sample labelling, microarray hybridization and wash-
ing were performed according to the manufacturer's protocols.
Briefly, total RNA was reverse transcribed to double-stranded
cDNA, which was subsequently converted into cRNA labelled with
cyanine-3-CTP; the cRNA was hybridized onto the microarray. For
the Illumina microarray assay, only one probe was designed for each
mRNA transcript. Microarray analysis was performed by Personalbio
Co.
2.11 | Cell immunofluorescence
Bone marrow monocytes (10,000/well) were inoculated in a 48-well
plate. The cells were treated with PBS or 100 μmol/L (100 µM) CoCl2
for 24 hr, fixed with 4% paraformaldehyde, permeabilized with 0.5%
Triton X-100 and blocked in 5% bovine serum albumin. After incuba-
tion for one day with anti-CTSK antibody or anti-cardiotrophin-1 an-
tibody at 4°C, the cells were incubated with fluorescein-conjugated
anti-rabbit or anti-mouse antibody at 37°C for 1 hr. The nuclei were
stained with 10 μl DAPI (1:1,000 DAPI in PBS).
2.12 | Protein extraction and Western blot assay
Bone marrow monocytes were seeded into 60-mm dishes and incu-
bated with PBS or 100 μM CoCl2 for 24 hr. Mandibles were ground
to homogenate with a homogenizer (Tissuelyser-24, Jingxin) 7 days
postsurgery. The cells or mandible was washed three times with
PBS and then harvested in RIPA buffer with protease inhibitor on
ice. The supernatants were collected and stored at −80°C and then
centrifuged at 16099 g for 10 min at 4°C. Protein contents were
determined by a BCA protein assay kit (Thermo). Protein samples
were isolated by 6%–10% sodium dodecyl sulphate–polyacrylamide
gel electrophoresis (SDS-PAGE) and then transferred to a polyvi-
nylidene fluoride (PVDF) membrane (Sigma-Aldrich). The PVDF
membranes were incubated for 1 hour with 5% non-fat milk to block
non-specific binding. Then, they were incubated with primary an-
tibodies against HIF-1α, CT-1 and β-actin at 4°C overnight. After
washing three times with TBST for 10 min, the membranes were in-
cubated with HRP-conjugated anti-rabbit or anti-mouse antibody at
37°C for 1 hr followed by washing with TBST three times for 10 min.
TIAN et al. | 431

Then, the membranes were visualized by the super-signal West-
Pico chemiluminescent substrate (Thermo Scientific). The band in-
tensity was analysed by a Smart ChemTM Image Analysis System
(Sagecreation). The protein bands were scanned and quantified by
ImageJ software.
2.13 | von Kossa staining and double-labelling assay
To investigate the mineralization rate of the bone microenviron-
ment in Ctrl and Cko mice, we performed double-labelling experi-
ments. Six Ctrl and Cko mice were intraperitoneally injected with
60 mg/kg calcein (Sigma-Aldrich). After a week, the mice were
injected with 60 mg/kg xylenol orange (Sigma-Aldrich). After
the second injection overnight, all the mice were sacrificed. The
mandible was completely separated, fixed in 4% paraformalde-
hyde for one day, and then dehydrated in a gradient of 50%, 75%,
95% and 100% ethanol for 30 min. The mandible was embedded
in Technovit 7,200 VLC resin (EXAKT, Germany) and sectioned
with a diamond band saw cutting system (EXAKT). The sample
was then stained with DAPI, diluted 1:800. For the von Kossa
staining assay, mandibular sections were placed in 1% silver ni-
trate (Sigma-Aldrich), irradiated with UV for 45 min, and stained
with 0.1% Kernechtrot (Sigma-Aldrich). Images were taken with a
microscope, and xylenol and calcein labelling was analysed with
ImageJ software.
2.14 | Enzyme-linked immunosorbent assay (ELISA)
Cko or Ctrl group BMMs were seeded into a 6-well plate and induced
to differentiate into osteoclasts with RANKL and M-CSF. After that,
the cells were washed twice with PBS, 2 ml of serum-free DMEM
culture medium was added, and the cells were cultured for 48 hr
to obtain the cell culture supernatant. The supernatant was centri-
fuged, and CT-1 was detected by "sandwich" ELISA (Ready-Set-Go
ELISA and Platinum ELISA, BOSTER) according to the manufactur-
er's instructions. A microplate reader (SYNERGY H1, BioTek) was
used to measure the absorbance of each well at 450 nm.
of serum-free DMEM culture medium was added, and the cells
were cultured for 48 hr to obtain the cell culture supernatant. The
conditioned medium (CM) was prepared mixing the previously
collected BMM supernatant with fresh bone marrow stem cell
(BMSC) medium in a 1:1 ratio. Bone marrow cells were isolated
from the femurs of 4-week-old C57BL/6 mice and cultured in
α-MEM basic medium containing 10% foetal bovine serum and 1%
penicillin–streptomycin. The medium was changed every 3 days,
and the cells were passaged when they reached 70%–80% conflu-
ence. Third-generation BMSCs obtained by the trypsin method
were seeded into 60-mm dishes and treated with Cko or Ctrl CM
or BMSC medium. To induce osteogenic differentiation, BMSCs
were cultured in osteogenic differentiation medium containing
Cko or Ctrl CM or BMSC medium and 10 mM β-glycerophosphate,
50 μg/ml ascorbic acid and 100 nM dexamethasone (Sigma-
Aldrich). Recombinant mouse CT-1 (Sigma-Aldrich) was added to
the medium in some groups. The osteogenic differentiation me-
dium was replaced each day.
2.16 | Transwell assay
A 24-well Transwell insert with an 8-μm pore size (Corning Costar)
was used to evaluate the migration of BMSCs under different CM.
BMSCs were trypsinized and resuspended in serum-free alpha-
DMEM 1 × 106/ml. One millilitre of Cko or Ctrl CM or serum-free
alpha-DMEM was added into the lower chambers, and 200 μl of a
BMSC suspension was added into the upper chambers. The contents
of the upper and lower wells were separated by a polycarbonate
membrane (8-μm pore size). Cells were allowed to migrate for 24 hr
at 37°C, after which the media were aspirated. The cells remaining
on the upper surface of the polycarbonate membrane were removed
with a cotton swab, and the cells that migrated to the lower surface
were stained with crystal violet (Beyotime, China) for 15 min. The
cells were counted under an inverted microscope by two researchers
separately. The average numbers of migrated cells were determined
by counting the cells.
2.17 | Cell counting Kit 8 assay

2.15 | Conditioned medium and osteogenic
differentiation
Cko or Ctrl group BMMs were seeded into a 6-well plate and
induced to differentiate into osteoclasts with RANKL and
M-CSF. After that, the cells were washed twice with PBS, 2 ml
Cell counting kit 8 (CCK-8, CK04, DOJINDO) reagent was used to
determine the growth capacity of BMSCs exposed to Cko or Ctrl
CM. Cells (5,000/well) were seeded in 96-well plates, and 10 μl of
CCK-8 solution was added to each well. After 12, 24, and 48 hr of
incubation, a microplate reader (SYNERGY H1, BioTek) was used to
measure the absorbance of each well at 450 nm.

F I G U R E 1 Mandibular osteotomy repair and the expression of HIF-1α in osteoclasts (a) A pattern diagram of mandibular osteotomy. (b)
Representative images of micro-CT. (c) The expression of tnfrsf11a, csf1r, ctsk, trap, mmp9, hif-1α and vegf mRNA in mandibular tissue after
sham surgery at 3, 7 and 14 days postsurgery (n = 3 each, one-way analysis of variance test). (d) Representative images of Western blot:
HIF-1α protein expression and analysis (n = 3 each, one-way analysis of variance test). (e) Decalcified mandibular bone stained with Trap in
the sham group and ps7d group. (f) Fluorescent immunohistochemistry of CTSK (green) and HIF-1α (red) 7 days postsurgery. Bars = 100 μm.
*p < .05, **p < .01. Data represent the mean ± SD [Colour figure can be viewed at wileyonlinelibrary.com]
432 | TIAN et al.
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434 | TIAN et al.

F I G U R E 2 Osteoclasts differentiated from Ctrl or Cko mouse BMMs. (a) The expression of ctsk, mmp9 and hif-1α mRNA in osteoclasts
differentiated from Ctrl or Cko mouse BMMs. (Cko mice = 6, Ctrl mice = 6, one-way analysis of variance test). (b) The expression of CTSK,
MMP9 and HIF-1α protein in osteoclasts differentiated from Ctrl or Cko mouse BMMs. Representative images of Western blot: CTSK,
MMP9 and HIF-1α protein expression and analysis. (c) The staining of Trap, HIF-1α, phalloidin and toluidine blue in osteoclasts differentiated
from Ctrl or Cko mouse BMMs. Images of bone absorption. (Cko mice = 6, Ctrl mice = 6, one-way analysis of variance test) (d) Statistical
analysis of the number of osteoclasts in the area of Trap, HIF-1α, phalloidin, toluidine blue and bone absorption. Bars = 100 μm. *, p < .05, **,
p < .01. Data represent the mean ± SD [Colour figure can be viewed at wileyonlinelibrary.com]

2.18 | Wound scratch assay
Cko or Ctrl CM was used in a wound scratch assay to test the mi-
gration ability of BMSCs. A straight line was drawn on the culture
disc using a 200-μl tip, and the cells were washed with PBS three
times. Then, BMSCs were cultured in Cko or Ctrl CM or αMEM me-
dium at 37°C for 24 hr in an incubator. Images were taken by a ZEISS
Axiocam microscope. The changes in the scratch area were deter-
mined by ImageJ.
2.19 | Alkaline phosphatase staining and Alizarin
red staining
Seven days after inducing osteogenic differentiation, the cells were
fixed in 4% PFA for 1 hr, rinsed with distilled water and stained with
an ALP staining kit (Sangon) following the manufacturer's instruc-
tions. The cells were incubated with ALP staining solution at 37°C
for 12 hr and then rinsed with distilled water three times. Fourteen
days after the induction of osteogenic differentiation, the cells
were fixed in 4% PFA for 1 hour and washed with distilled water.
The mineralized nodules were stained with 40 mm Alizarin red solu-
tion (Sigma-Aldrich) for 20 min and then washed with distilled water
three times. Images were taken with a stereomicroscope (Zeiss) and
analysed with ImageJ.
The mRNA expression of the osteoclast-related genes tnfrsf11a,
csf1r, ctsk, trap and mmp9 in the surgery group was significantly
increased, particularly at 7 days postsurgery (ps7d group), compared
with that in the sham group (Figure 1c), suggesting that the repair
process requires the participation of osteoclasts. Moreover, HIF-1α
and vegf mRNA levels and HIF-1α protein levels were increased in
the ps7d group (Figure 1c,d). This finding confirmed that the repair
process occurred in a hypoxic environment. As shown in Figure 1e,
more multinucleated and Trap-positive cells adhered to the bone
stump in the ps7d group than in the sham group. Moreover, the
CTSK and HIF-1α co-stained cells were significantly increased com-
pared with those in the sham group (Figure 1f and Appendix Figures
S1b), which indicated that osteoclast HIF-1α might play a key role in
bone gap repair. Thus, we generated Ctrl and Cko mice by intercross-
ing mice homozygous for a floxed HIF-1α allele with mice harbouring
Cre in the cathepsin K locus. We observed that the multinucleated
and Trap-positive cells adhered to the bone stump in Cko mice, and
the CTSK and HIF-1α co-stained cells were significantly decreased
in the Cko mice compared with the Ctrl mice, as shown in Appendix
Figure S1c,d. These results indicated that HIF-1α-expressing osteo-
clasts were recruited to the site of mandibular osteotomy.
3.2 | HIF-1α-knockout primary osteoclasts inhibited
bone resorption

Bone marrow monocytes extracted from the bone marrow of Cko

2.20 | Statistical analysis
All experiments in this study were repeated at least three times, and
statistical analyses were performed using SPSS 20.0 and GraphPad
Prism 7.0. All quantitative data are expressed as the mean ± stand-
ard deviation in each case. Differences between groups were ana-
lysed by one-way analysis of variance (ANOVA) and Tukey analysis,
and p < .05 was considered statistically significant.
3 | R E S U LT S
3.1 | HIF-1α-expressing osteoclasts were recruited
to the site of mandibular osteotomy repair
Mandibular osteotomy was performed using a 1-mm-diameter drill
bit (Figure 1a, Appendix Figure S1a). Three-dimensional reconstruc-
tions and cross-sections revealed the gap, as indicated in Figure 1b.
and Ctrl mice were induced to differentiate into osteoclasts by
M-CSF and RANKL. CoCl2, a chemical inducer of HIF-1α, was used
to induce HIF-1α DNA-binding activity to mimic hypoxia. To deter-
mine the function of HIF-1α in osteoclasts during osteoclastogen-
esis and bone resorption, HIF-1α expression was increased with
CoCl2, and PBS was used as a negative control. The transcription
of osteoclast-related genes (ctsk and mmp9) and HIF-1α was quan-
tified by real-time PCR and Western blot analyses (Figure 2a,b).
Regardless of the presence of CoCl2, the gene and protein expres-
sion and the number of Cko primary osteoclasts were lower than
those of Ctrl primary osteoclasts. More multinucleated and Trap-
positive cells were found in the Ctrl group than in the Cko group
(Figure 2c). CoCl2 treatment enhanced osteoclast differentiation in
the Ctrl group but not in the Cko group. As expected, the Cko group
presented decreased areas of phalloidin, toluidine blue and HIF-1α
staining and decreased bone resorption (Figure 2c,d). These results
confirm that HIF-1α plays a key role in osteoclast-mediated bone
resorption in vitro.
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F I G U R E 3 Comparison of histology and morphology during bone healing between Ctrl and Cko mice. (a) X-ray and three-dimensional
reconstruction of Ctrl and Cko mice 1, 3 and 5 weeks postsurgery. (b) Statistical analysis of histomorphometric values including trabecular
thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular bone volume fraction (BV/TV) and trabecular number (Tb.N) (Cko mice = 6, Ctrl
mice = 6, one-way analysis of variance test). (c) The staining of HE, ALP and Trap between Ctrl and Cko mice. Immunohistochemistry with
anti-Ocn antibody, von Kossa staining and xylenol orange and calcein double-labelling assay of Ctrl and Cko mice. Statistical analysis of new
bone area. Bars = 100 μm. *, p < .05, **, p < .01. Data represent the mean ± SD [Colour figure can be viewed at wileyonlinelibrary.com]

3.3 | Bone repair was delayed in osteoclast-specific 3.5 | HIF-1α-knockout primary osteoclasts inhibited
HIF-1α-knockout mice osteogenesis via the coupling factor CT-1

To test the influence of HIF-1α in osteoclasts during bone repair in vivo,
we compared the Cko and Ctrl mice at different times by 3D recon-
struction and X-ray radiography. The bone gap was comparatively nar-
rower in Ctrl mice, and almost complete closure was found in the Ctrl
mice at 5 weeks postsurgery, as demonstrated in the sagittal and lateral
views (Figure 3a). The trabecular bone volume fraction (BV/TV) and
trabecular thickness (Tb.Th) were significantly lower in the Cko mice
than in the Ctrl mice. Higher trabecular separation (Tb.Sp) was found
in the Cko mice than in the Ctrl mice. The trabecular number (Tb.N)
in the two groups of mice was not significantly different (Figure 3a).
Moreover, HE staining showed significantly delayed bone repair in the
Cko group (Figure 3b). The ALP-positive area in the Cko group was
smaller than that in the Ctrl group (Figure 3b). Immunohistochemistry
with anti-Ocn antibody indicated that the ossification-related protein
Ocn was expressed at low levels in the Cko group (Figure 3c), and
von Kossa staining demonstrated a high level of mineralization in the
bone gap in the Ctrl mice (Figure 3d). The double-labelling assay dem-
onstrated a higher bone formation rate in Ctrl mice (Figure 3d). The
degree and speed of bone healing showed clear differences between
the Cko and Ctrl mice. These results indicate that HIF-1α expression in
osteoclasts is important for the bone repair process.
3.4 | HIF-1α enhanced the osteoclast stimulation of
BMSC differentiation
Because HIF-1α expression in osteoclasts is important for the bone
repair process, we next aimed to investigate how HIF-1α regulates
osteogenesis or even the crosstalk between osteoclasts and os-
teoblasts. Thus, we used CM from osteoclasts to culture BMSCs.
BMSC cultures were exposed to supernatants from Cko and Ctrl
primary osteoclasts as CMs and alpha-MEM as a negative control.
CCK-8 assays showed that the CMs did not affect BMSC prolifera-
tion (Figure 4a). Both Transwell and wound scratch assays showed
that the Ctrl supernatant slightly enhanced the migration of BMSCs
compared with that obtained with the Cko supernatant (Figure 4b,c).
Osteogenic differentiation experiments revealed that the transcrip-
tion of osteoblast differentiation-related genes (alp and runx2) was
increased in the presence of the Ctrl supernatant (Figure 4d). ALP
and Alizarin red staining showed that the Cko supernatant did not
improve the osteogenic differentiation ability (Figure 4b). These
results confirm that HIF-1α influences crosstalk in primary induced
osteoclasts.
To determine the key player in the mechanism by which HIF-1α influ-
ences the crosstalk between osteoclasts and osteoblasts, we ana-
lysed Cko and Ctrl primary osteoclasts by mRNA sequencing. We
found that the expression of osteoclast-related genes, such as Traf6,
Ocstamp, Mmp9, Ctsk and Dcstamp, was decreased in the Cko group
(Figure 5a) and that HIF-1α affected the expression of these genes.
Moreover, Ctf1 (the gene that encodes CT-1) was significantly de-
creased in Cko osteoclasts. Therefore, CT-1 was detected by immu-
nofluorescence, real-time PCR and Western blotting (Figure 5b-d).
Interestingly, the CT-1-stained area and the mRNA and protein ex-
pression levels were consistent with HIF-1α expression, and HIF-1α
expression in osteoclasts was correlated with CT-1 expression. To
confirm that HIF-1α not only influenced the expression of CT-1 in
osteoclasts but also impacted the secretion of CT-1 by osteoclasts,
we measured the expression of CT-1 in Ctrl and Cko osteoclast su-
pernatants by ELISA. As expected, CT-1 was decreased in the Cko
group (Figure 5e). These results suggest that HIF-1α expression in
osteoclasts influences CT-1 expression. To determine whether HIF-
1α-knockout primary osteoclasts inhibit bone repair through the
decreased secretion of CT-1, the level of CT-1 in the postsurgery
mandible tissues of 3-week-old Cko and Ctrl mice was detected by
immunofluorescence (Figure 5f). CT-1 expression was higher in the
Ctrl group than the Cko group. Thus, we used 0/2.5/5/10 µg/ml re-
combinant CT-1 to induce the osteogenic differentiation of BMSCs
and found that 2.5/5/10 µg/ml recombinant CT-1 improved osteo-
genic differentiation and that 5 µg was the best concentration of
CT-1 for osteogenic differentiation (Appendix Figure S2b,c). We
used siCT-1 (sictf-2 is included in Appendix Figure S2a) to silence
CT-1 in Ctrl primary osteoclasts. We added siCT-1 supernatant or
5 µg/ml recombinant CT-1 to BMSC medium for osteogenic differ-
entiation (Figure 5g). We found that osteogenesis was highest in the
Ctrl supernatant + CT-1 group and lowest in the Cko supernatant
group. Moreover, CT-1 could rescue the osteogenesis induced by
HIF-1α knockout in osteoclasts. These results confirm that HIF-1α
influences osteogenesis in osteoclasts via the coupling factor CT-1.
4 | D I S CU S S I O N
Orthodontic-orthognathic combination therapy is a classic method
for the treatment of bone malocclusion (Patel & Novia, 2007), but this
treatment invariably causes iatrogenic bone trauma to the mandi-
ble. Hypoxia is one of the characteristics of the microenvironmental
TIAN et al. | 437

F I G U R E 4 Supernatants from Ctrl or Cko mouse osteoclasts co-cultured with BMSCs. (a) CCK-8 assay was used to test the proliferation
of BMSCs cultured in supernatants from Ctrl or Cko mouse osteoclasts, with α-MEM used as a control (n = 3, Tukey analysis). (b) Transwell
migration assay (24 hr at 37°C) of BMSCs cultured in Cko or Ctrl CM or α-MEM as the control. Wound scratch assay of BMSCs cultured
in Cko or Ctrl CM or α-MEM as the control for 0 hr and 24 hr. ALP staining after 7 days and Alizarin red staining after 14 days of BMSCs
isolated from mice and cultured in osteogenic differentiation medium containing Cko or Ctrl CM. (c) Statistical analysis of BMSC numbers
from the Transwell assay and migration numbers from the wound scratch assay and the ALP and Alizarin red staining area (n = 3, Tukey
analysis). (d) The expression of alp and runx2 mRNA in BMSCs differentiated from Ctrl or Cko mouse BMMs and cultured in osteogenic
differentiation medium containing Cko or Ctrl CM or α-MEM as the control. (n = 3, Tukey analysis) Bars = 100 μm. *, p < .05, **, p < .01. Data
represent the mean ± SD [Colour figure can be viewed at wileyonlinelibrary.com]

changes observed after bone trauma (Dehne & Brüne, 2009; Loi
et al., 2016). As the injured site gradually becomes hypoxic (Maes
et al., 2012; Sadiku & Walmsley, 2019), the partial oxygen pres-
sure in the centre of the fracture can decrease to 0 mm Hg (Miclau
et al., 2017), and our study indicates increased HIF-1α expression
and recruitment of HIF-1α-expressing osteoclasts to the injured site.
Bone repair is a complex process that is mainly modulated by os-
teoclasts and osteoblasts (Epari et al., 2008) and is mediated by two
mechanisms: cartilage osteogenesis and intramembrane osteogen-
esis (Fang & Hall, 1997; Marsell & Einhorn, 2011). The craniofacial
438 | TIAN et al.

F I G U R E 5 CT-1 expression decreased in HIF-1α-knockout primary osteoclasts in vitro and in vivo. (a) Gene heat map of the mRNA
sequence of osteoclasts differentiated from BMMs from Cko or Ctrl mice. (b) Fluorescent immunohistochemistry of CTSK (green) and CT-1
(red) in osteoclasts differentiated from Ctrl or Cko mouse BMMs. (c) The expression of CT-1 mRNA in osteoclasts differentiated from Ctrl
or Cko mouse BMMs (n = 3, Tukey analysis). (d-e) The expression of CT-1 protein in osteoclasts induced from Ctrl or Cko mouse BMMs.
Representative images of Western blot: CT-1 protein expression and analysis (n = 3, Tukey analysis). (f) Fluorescent immunohistochemistry
of CT-1 (green) between Ctrl and Cko mice 3 weeks postsurgery. (g) Alizarin red staining of BMSCs isolated from mice and cultured for
14 days in osteogenic differentiation medium containing Cko or Ctrl CM supplemented with recombinant CT-1 or siCT-1. Bars = 100 μm. *,
p < .05, **, p < .01. Data represent the mean ± SD [Colour figure can be viewed at wileyonlinelibrary.com]
TIAN et al.
bone is mainly repaired by intramembranous osteogenesis, which is
a relatively simple osteogenic model. Without the involvement of
chondrocytes, the newly formed bone is directly differentiated from
mesenchymal stem cells into osteoblasts (Runyan & Gabrick, 2017).
Thus, we selected mandibular osteotomy, which involves the partial
removal of bone tissues neighbouring the incisor, as a research model
to investigate the hypoxic environment, bone repair and crosstalk
between osteoclasts and osteoblasts (Granström & Nilsson, 1987;
Ransom et al., 2018).
Bone repair occurs in a hypoxic environment with increased
HIF-1α expression (Semenza, 2012; Wang et al., 2007), and many
osteoclasts participate in this process (Arnett & Orriss, 2018;
Stegen et al., 2016). According to the presence of HIF-1α-positive
osteoclasts in the bone gap after osteotomy, we hypothesized that
osteoclasts might play a crucial role in this process. In vitro exper-
iments revealed that HIF-1α not only enhanced osteoclast-related
gene and protein expression but also the number of TRAP-positive
osteoclasts, which was consistent with the results from other stud-
ies (Shirakura et al., 2010). To determine the function of HIF-1α in
osteoclasts during bone repair, we bred osteoclast-specific HIF-1α-
conditional-knockout(Cko) mice. Primary HIF-1α-knockout osteo-
clasts showed reduced differentiation and bone absorption. There is
evidence that HIF-1α is required for osteoclast activation (Hadi et al.,
2015; Hinoi et al., 2012; Tang et al., 2019) and bone loss in osteopo-
rosis (Miyauchi et al., 2013; Tando et al., 2016). Cko mice showed
obvious delayed bone repair during the mandibular osteotomy re-
pair process compared with Ctrl mice. Osteoclast-mediated bone
resorption is the initial stage of bone reconstruction, and the inter-
action between osteoclasts and osteoblasts is critical during bone
repair (Araldi & Schipani, 2010; Cho et al., 2010; Huang et al., 2016).
However, the mechanism by which HIF-1α influences the function of
osteoclasts in osteogenesis needs further research. Thus, a review
of the literature and RNA microarray analysis revealed that CT-1,
which is a gp130-signalling cytokine belonging to the IL-6 super-
family, might be a key hub linking HIF-1α with osteoclast function.
Our previous work showed that IL-6 can enhance osteoclastogenesis
(Wu et al., 2017) and that CT-1 can stimulate osteoclast differentia-
tion in vitro (Richards et al., 2000). Furthermore, despite their high
osteoclast numbers, CT-1-deficient mice showed markedly lower
absorptive activity and clearly decreased bone formation, which
suggested that CT-1 can promote osteoblast differentiation and os-
teogenesis both in vitro and in vivo (Walker et al., 2008). In addition,
HIF-1α is involved in the hypoxia-induced upregulation of CT-1 in
heart diseases (Ateghang et al., 2006; Ng et al., 2010), and its over-
expression induces CT-1 promoter activity via functional hypoxia
response elements (Robador et al., 2011). Our results confirmed that
HIF-1α is involved in the upregulation of CT-1 during bone repair and
that osteoclasts can secrete more CT-1 under CoCl2 mimic hypoxia
conditions accompanied by markedly higher absorptive activity.
Immunofluorescence detection of CT-1 expression in bone re-
pair tissues showed that the expression of CT-1 was higher in the
Ctrl group than in the Cko group, which indicated the existence of
a relationship among HIF-1α, CT-1 and bone repair. Because CT-1
| 439
is a coupling factor that also promotes osteoblast differentiation
(Walker et al., 2008, 2010), we used a conditional co-culture of os-
teoclasts and BMSCs to determine whether primary osteoclasts of
Cko mice influence the osteogenesis of BMSCs via CT-1 in compari-
son with the Ctrl group. Our study suggests that knocking out HIF-1α
in primary osteoclasts can inhibit bone repair by decreasing the se-
cretion of CT-1. In addition, although the conditional co-culture did
not influence proliferation, it did enhance the osteogenesis abilities
of BMSCs. Knocking out HIF-1α or CT-1 exerted a similar effect on
the crosstalk between osteoclasts and osteoblasts. Our results in-
dicated that the recombination of CT-1 could rescue the function
of HIF-1α-knockout osteoclasts on the crosstalk with osteoblasts.
In summary, this study demonstrated that osteoclasts expressing
HIF-1α are recruited after mandibular osteotomy and that secreted
CT-1 acts as a coupling factor that stimulates the osteogenic dif-
ferentiation of BMSCs. Here, we have confirmed that HIF-1α is re-
quired for bone absorption, and HIF-1α could stimulate osteoclasts
to secrete more CT-1 and promote the osteogenic response in an
osteotomy model of bone repair. Thus, HIF-1α in osteoclasts could
be targeted to induce bone absorption in diseases such as osteo-
petrosis, and HIF-1α could augment mandibular bone regeneration
after injury, such as mandibular fracture. In addition, while we did
not used recombinant CT-1 in vivo in this study, we will perform this
rescue experiment in mandibular fractures in the future.
AC K N OW L E D G E M E N T S
Junying LI critically revised the manuscript for grammar. Financial
support was provided by the National Natural Science Foundation of
China (Grant no. 81670961) and Technology Committee Foundation
of Shanghai（Grant no. 20Y11904000）.
C O N FL I C T O F I N T E R E S T S
None to declare.
AU T H O R C O N T R I B U T I O N S
Yuanye Tian: Conceptualization; Data curation; Formal analysis;
Investigation; Writing-original draft; Writing-review & editing. qi
shao: Data curation; Methodology; Resources; Writing-original
draft; Writing-review & editing. Yi Tang: Investigation; Methodology;
Project administration. Xinzhao Li: Data curation; Formal analysis.
Xin Qi: Formal analysis; Methodology. Runyang Jiang: Methodology;
Resources. Yi Liang: Writing-original draft; Writing-review & editing.
Feiwu Kang: Funding acquisition; Investigation; Project administra-
tion; Writing-review & editing.
AU T H O R C O N T R I B U T I O N S
Yuanye Tian and Qi Shao contributed to the conception and design
of the study, data acquisition, analysis and interpretation and drafted
and critically revised the manuscript. Yi Tang contributed to the con-
ception and design of the study, sample processing, data acquisi-
tion, analysis and interpretation, and drafted and critically revised
the manuscript. Xin Qi, Xinzhao Li, and Runyang Jiang contributed
to sample processing and data acquisition. Yi Liang contributed to
440 | TIAN et al.
data interpretation and drafted and critically revised the manuscript.
Feiwu Kang contributed to the conception and design of the study,
data analysis, and interpretation and critically revised the manu-
script. All authors gave final approval and agreed to be accountable
for all aspects of the work.
PEER REVIEW
The peer review history for this article is available at https://publo​
ns.com/publo​n/10.1111/odi.13745.
ORCID
Yuanye Tian https://orcid.org/0000-0003-3671-5852
Feiwu Kang https://orcid.org/0000-0003-4973-3857
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S U P P O R T I N G I N FO R M AT I O N
Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: Tian Y, Shao Q, Tang Y, et al. HIF-1α
regulates osteoclast activation and mediates osteogenesis
during mandibular bone repair via CT-1. Oral Dis.
2022;28:428–441. https://doi.org/10.1111/odi.13745