Journal of Cranio-Maxillo-Facial Surgery 49 (2021) 738e747

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Journal of Cranio-Maxillo-Facial Surgery

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Lineage-associated connexin 43 expression in bisphosphonate-

exposed rat bones
Raimund H.M. Preidl c, *, Kerstin Amann b, Manuel Weber c, Martin Schiller d,
Manuela Ringler d, Jutta Ries e, Friedrich W. Neukam f, Marco Kesting g,
Carol-Immanuel Geppert h, Falk Wehrhan a
a
Specialist in Oral and Maxillofacial Surgery, Department of Oral and Maxillofacial Surgery, University of Erlangen-Nuremberg, Glückstraße 11, 91056,
Erlangen, Germany
b
Head of Nephropathology, University of Erlangen-Nuremberg, Glückstraße 11, 91056, Erlangen, Germany
c
Resident, Department of Oral and Maxillofacial Surgery, University of Erlangen-Nuremberg, Glückstraße 11, 91056, Erlangen, Germany
d
Doctoral Students, Department of Oral and Maxillofacial Surgery, University of Erlangen-Nuremberg, Glückstraße 11, 91056, Erlangen, Germany
e
Department of Oral and Maxillofacial Surgery, University of Erlangen-Nuremberg, Glückstraße 11, 91056, Erlangen, Germany
f
Former Head of Department of Oral and Maxillofacial Surgery, University of Erlangen-Nuremberg, Glückstraße 11, 91056, Erlangen, Germany
g
Head of Department of Oral and Maxillofacial Surgery, University of Erlangen-Nuremberg, Glückstraße 11, 91056, Erlangen, Germany
h
Specialist in Pathology, University of Erlangen-Nuremberg, Glückstraße 11, 91056, Erlangen, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Expression of signaling proteins in bone cells depends on their embryological mesoderm-derived (e.g.
Paper received 14 February 2020 tibia) or cranial neural crest (CNC)-derived (e.g. jaw) origin. Connexin 43 (Cx43) is a gap junction protein
Received in revised form that plays an essential role in the mode of action of bisphosphonates (BP). This study aimed to investigate
19 November 2020
Cx43 expression and the influence of BP application on mesoderm- and CNC-derived bone.
Accepted 14 February 2021
Using a rat model, molar extraction and tibia osteotomy with (Group 4) or without (Group 3) pre-
Available online 18 February 2021
vious BP application was performed. Untreated (Group 1) and animals selectively treated with BPs
(Group 2) served as controls. Cx43 expression was immunohistochemically determined 12 and 16 weeks
Keywords:
Medication-related osteonecrosis of the jaw
postoperatively via a labeling index.
(MRONJ) Cx43 expression in CNC-derived bone was significantly higher compared with mesodermal bone. BP
Bisphosphonate application decreased Cx43 expression; however, detected expression levels were still higher in jawbone
Jawbone (Group 2 tibia vs jaw: 5.83 ± 5.06 vs 23.52 ± 6.42; p ¼ 0.007). During bone healing after surgical
intervention (Group 3) there were no expression differences between tibia and jawbone. BP treatment
prior to surgery resulted in significantly lower Cx43 expression in CNC-derived compared with tibia bone
(Group 4 tibia vs jaw: 56.84 ± 15.57 vs 16.40 ± 5.66; p < 0.01).
Increased Cx43 expression in jaw compared with tibia bone is in line with their embryological ori-
gins. A significant Cx43 suppression in jawbone after BP application and surgery might contribute to the
selectively altered osseous turnover and development of MRONJ in CNC-derived bone.
© 2021 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights
reserved.

1. Introduction osteonecrosis of the jaw (BRONJ) in 2003, the number of affected

Bisphosphonates (BP) are frequently applied in patients
suffering from bone metastases or osteoporosis because of their
high affinity to bone and their ability to inhibit osteoclastic bone
resorption. Since the first description of bisphosphonate-related
patients presenting with necrotic jawbone, often associated with
mucosal swelling, erythema, or even abscesses, continued to in-
crease (Marx 2003). In the meantime, additional antiresorptive
drugs that interfere with bone turnover, such as denosumab, have
been shown to cause identical necrotic jaw lesions to those

* Corresponding author.
E-mail address: raimund.preidl@uk-erlangen.de (R.H.M. Preidl).

https://doi.org/10.1016/j.jcms.2021.02.010
1010-5182/© 2021 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
R.H.M. Preidl, K. Amann, M. Weber et al.
described in BRONJ. Therefore, this disease pattern d involving
exposed necrotic jawbone for a period of >8 weeks and a patient
history of antiresorptive drug treatment in the absence of radio-
therapy d is summarized as medication-related osteonecrosis of
the jaw (MRONJ) or antiresorptive agent-related osteonecrosis of
the jaw (ARONJ) (Ruggiero et al., 2014; Yoneda et al., 2017).
In the current literature, numerous clinical and laboratory in-
vestigations targeting a pathophysiological understanding of the
disease and the development of treatment strategies have been
published. However, the processes and reasons leading to osteo-
necrosis occurring exclusively in the jawbone are still not
completely understood. Earlier theories of an avascular necrosis
initiating BRONJ seem to have been revised (Hansen et al., 2006).
Another theory is based on the unique anatomical and microbio-
logical characteristics of the jawbone, resulting in direct exposure
to the oral bacterial flora after mucosal damage and therefore
leading to local inflammation (Yoneda et al. 2017).
From an embryological point of view, jawbone cells are cranial
neural crest (CNC)-derived and exhibit intramembranous bone
formation, whereas bone cells of the axial skeleton, such as the
ilium or the tibia, are derived from the mesoderm and undergo
endochondral as well as intramembranous bone formation. These
site-specific differences go along with different characteristics in
terms of osteoblastic and osteoclastic differentiation, and consec-
utive bone formation in the jaw (Lee et al. 2015). Although the
bones are comparable in phenotypic structure, numerous regula-
tory genes and proteins in bone tissue show different expression
patterns in CNC-derived bone. In a previous publication, our group
was able to demonstrate that homeobox gene Msx-1 was decreased
by sevenfold in jawbone affected by BRONJ compared with healthy
jawbone without BP exposure, illustrating impaired regenerative
capacities (Wehrhan et al. 2011).
The process of bone remodeling is an important component of
skeleton integrity as well as renewal, and relies on the orchestrated
activation and coupling of osteoclastic and osteoblastic cells,
especially within a disease environment. Osteocytes embedded
inside the bone matrix form a network, with dendritic processes
connecting neighboring osteocytes but also osteoclasts and osteo-
blasts on the bone surface (Bonewald 2005). Within this network,
intercellular communication via connecting gap junctions, such as
connexin 43 (Cx43), located on osteocytes and osteoblasts, have
been found to be essential for osteocytic regulation and consecutive
bone remodeling (George et al. 2018). Cx43, being the most highly
expressed connexin in bone, is essential for direct intercellular
coupling among bone cells regulating osteoblast differentiation and
bone homeostasis (Sadr-Eshkevari et al. 2014; Xu et al. 2015; Uribe
et al. 2018). A lack of Cx43 is known to cause generalized osteoblast
dysfunction, resulting in decreased mineralization and skull ab-
normalities (Lecanda et al. 2000). Furthermore, Cx43-deficient
bone tissue presents increased osteocyte apoptosis, enhanced
osteoclast recruitment to the surface, and bony defects (Bivi et al.
2012).
With regard to lineage-specific protein expressions in bones,
and in consideration of Cx43 being highly expressed in CNC-
derived tissue, as well as the site-specific occurrence of MRONJ,
the aim of this study was to investigate the influence of
bisphosphonates on the expression of Cx43 in mesenchymal and
CNC-derived bone compared with an unaltered physiological sit-
uation. Bearing in mind the essential role of Cx43 expression during
bone healing, our study also aimed to investigate and compare
changes in Cx43 expression in regular and BP- influenced bone
healing situations, in both lineages.
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Journal of Cranio-Maxillo-Facial Surgery 49 (2021) 738e747
2. Materials and methods
2.1. Animals
Adult Wistar rats (Charles River Wiga, Sulzfeld, Germany), aged
between 6 and 8 months at the time of experimental use, were
maintained in the central animal facility of the university under
specific pathogen-free conditions. This study was carried out in
accordance with international guidelines and the guidelines of the
Government’s Animal Care and Use Committee (AZ 54-2532.1-3/
09). Rats were fed a regular diet with water ad libitum, and were
maintained on a 12-hour light/dark cycle. Animals were randomly
divided into four groups, with a total of ten animals per group d
five for each investigational time point (Fig. 1). G1 consisted of
untreated controls. In G2, rats received intraperitoneal injections of
zoledronate 40 mg/kg bodyweight (Zometa®; Novartis Pharma,
Basel, Switzerland) d one injection per week for 8 weeks. In G3,
rats underwent unilateral extraction of the mandibular first molar,
without plastic wound closure, and an osteotomy of the tibial bone
in the midsection, using a standardized molding cutter. This was
immediately followed by rigid osteosynthesis with an eight-hole,
2.0 miniplate and four screws d two on each side of the fracture
(Stryker, Kalamazoo, Michigan, USA). Wound closure was per-
formed with continuous sutures (Vicryl 4-0; Ethicon, Norderstedt,
Germany) after closure of the periostium. In G4, rats received
zoledronate for 8 weeks prior to the surgical procedure described in
G3. Tooth extraction and tibial bone osteotomy, with immediate
osteosynthesis, were conducted under general anesthesia using
100 mg/kg of ketamine hydrochloride (Parke-Davis, Berlin, Ger-
many) and 2.5 mg/kg of Xylacin (Bayer, Leverkusen, Germany) via
intraperitoneal injection. Postoperative analgesia was conducted
with 2 mg/kg i.p. buprenorphin (Temgesic; Essex Pharma, Munich,
Germany) on the first and second postoperative day, and beyond if
necessary.
Animals were sacrificed after 12 weeks (five out of ten in each
group) or 16 weeks (the remaining animals in each group) using an
overdose of anesthetic. The bone samples (mandible and tibia)
were harvested and immediately stored in 4% formalin solution. In
Groups 3 and 4, osteosynthesis was removed prior to histological
processing.
2.2. Immunohistochemical staining
Samples were decalcified in 10% EDTA (pH 7.4) in an ultrasonic
bath before embedding in paraffin. After serial sectioning in a
microtome and dewaxing in graded alcohol, immunohistochemical
staining was performed using the alkaline-phosphatase-anti-
alkaline phosphatase (APAAP-method) according to the manufac-
turer’s instructions (Dako EnVision HRP Kit K5007, Glostrup,
Denmark). Connexin 43 was targeted with a polyclonal rabbit
antibody to anti-rat connexin 43 (anti-connexin 43, ab11370;
abcam, Cambridge, UK), 1:100 for jaw and tibial bone, respectively.
Antibodies were incubated with tissue sections for 35 minutes
before secondary antibody was applied.
2.3. Semiquantitative immunohistochemical analysis
Sections used in this analysis were digitalized using the ‘whole-
slide imaging’ method described in a previous study (Weber et al.
2015). All scanned samples were virtually microscoped on PC
(Panoramic MIRAX viewer; Zeiss, Jena, Germany). Three visual
fields per section at a magnification of 200 were selected for each
R.H.M. Preidl, K. Amann, M. Weber et al. Journal of Cranio-Maxillo-Facial Surgery 49 (2021) 738e747

Fig. 1. Experimental setting for this BP-treatment animal model. Rats were allocated to four different groups, with Group 1 as untreated controls and Group 2 animals receiving BP
treatment via weekly zoledronate application, intraperitoneally for 8 weeks. Group 3 rats underwent extraction of the mandibular first molar and osteotomy of one tibial bone,
followed by immediate rigid osteosynthesis. Group 4 animals were pretreated with BP prior to extraction of the mandibular first molar and osteotomy of one tibial bone with
osteosynthesis. 12 and 16 weeks after the respective interventions all animals in Groups 3 and 4 were sacrificed and bone tissue was fixed for immunohistochemical investigation.

sample. The visual fields of bone tissue were imported into Optimas 3. Results
6.5 (Media Cybernetics, Rockville, USA) to perform semi-
quantitative analysis, with selective counting of osteocytes within 3.1. General morphological and immunohistochemical
the cortical bone by three independent observers. In Groups 3 and 4 considerations
the visual fields were selected next to the defects in bone tissue.
Cx43-specific immunohistochemical staining was detected on
bone-associated cells, such as osteocytes and osteoblasts, as well as
2.4. Statistical analysis
on vascular endothelial cells in the selected regions of interest.
Positive staining was seen not only along the cell membrane but
The labeling index for each visual field (ratio: positive stained
also in the cytoplasm of the cells, visualizing newly formed Cx43
cells per total amount of cells per visual field) was calculated for
being transferred to the cell membrane. Aside from bone-specific
each timepoint, using the included number of animals in each
staining, Cx43 was also expressed in tooth-associated tissue, such
group and the respective tissue. Results were expressed as median
as the periodontium, the root channels, and the mucosal and sub-
(M), interquartile range (IR), and 75th as well as 25th percentiles in
mucosal layers surrounding the teeth. In the rat jaws, this was not
box plots, and as median ± standard deviation in Table 1. ANOVA
only observed on the molars but also on the long incisors of the
testing was conducted and p-values  0.05 were considered as
lower jaw, situated in the jaw corpus (Fig. 3). In tibial bone, Cx43
significant. All analyses were performed using SPSS 21.0 for Mac
expression was not only found in bone, but also in cells situated in
(IBM Inc., New York, USA).

Table 1
Data for all four investigated groups, with their respective time points, comparing CNC-derived jawbone with mesoderm-derived tibial bone (upper section), and the influence
of BP with and without surgical intervention on the respective tissues (lower section). Data are presented as median ±standard deviation.

Tibia Jaw Tibia Jaw p-value

Week 12 Week 12 p-value Week 16 Week 16

Group 1 10.01 ± 2.93 43.50 ± 14.36 p ¼ 0.01 12.60 ± 4.46 27.76 ± 10.37 p ¼ 0.01

Group 2 7.54 ± 9.29 12.06 ± 10.54 p ¼ 0.567 5.83 ± 5.06 23.52 ± 6.42 p ¼ 0.007

Group 3 31.35 ± 14.95 16.50 ± 13.37 p ¼ 0.154 32.54 ± 12.67 42.26 ± 15.02 p ¼ 0.484

Group 4 35.68 ± 20.30 34.99 ± 10.23 p ¼ 0.540 56.84 ± 15.57 16.40 ± 5.66 p < 0.01

Tibia Group 1 Group 2 Group 1 Group 2

Week 12 Week 12 Week 16 Week 16
10.01 ± 2.93 7.54 ± 9.29 p ¼ 0.392 12.60 ± 4.46 5.83 ± 5.06 p ¼ 0.065

Jaw Group 1 Group 2 Group 1 Group 2

Week 12 Week 12 Week 16 Week 16
43.50 ± 14.36 12.06 ± 10.54 p ¼ 0.007 27.76 ± 10.37 23.52 ± 6.42 p ¼ 0.178

Tibia Group 3 Group 4 Group 3 Group 4

Week 12 Week 12 Week 16 Week 16
31.35 ± 14.95 35.68 ± 20.30 p ¼ 0.469 32.54 ± 12.67 56.84 ± 15.57 p ¼ 0.033

Jaw Group 3 Group 4 Group 3 Group 4

Week 12 Week 12 Week 16 Week 16
16.50 ± 13.37 34.99 ± 10.23 p ¼ 0.026 42.26 ± 15.02 16.40 ± 5.66 p ¼ 0.005

740
R.H.M. Preidl, K. Amann, M. Weber et al.
Fig. 2. a) Boxplots presenting a significantly higher Cx43 expression in CNC-derived jawbone compared with mesoderm-derived tibial bone in the control Group 1. b) After
intraperitoneal BP application for 8 weeks, Cx43 expression in jawbone was not significantly different from that in tibial bone 12 weeks after the last application. Over subsequent
weeks Cx43 expression rose selectively in CNC-derived jawbone and expression was significantly higher 4 weeks later compared with tibial bone.
the marrow. In Groups 3 and 4, expression could also be detected in
the fibrous scars that developed around the bone and integrated
osteosynthesis. Additionally, the area within the former fracture
zone was characterized by increased staining intensity (Fig. 5).
3.2. Cx43 protein expression was significantly higher in CNC-
derived bone compared with mesoderm-derived bone after BP
application and with untreated controls
Immunohistochemical analysis revealed significantly higher
Cx43 expression rates in the jaw compared with the tibia at both
investigational timepoints in the untreated controls (p ¼ 0.01 for
each group) (Fig. 2a; Table 1). 16 weeks after cessation of BP
treatment, CNC-derived jawbone presented significantly higher
expression rates compared with mesodermal tibial bone (jawbone
G3 vs G4 at week 12: p ¼ 0.007) (Figures 2b and 3). However, when
comparing BP-treated animals in G2 with the untreated controls in
G1, BPs was shown to cause a general decrease in protein expres-
sion in both investigated types of bone (Figures 2a and 2b; Table 1).
3.3. Surgical intervention decreased Cx43 protein expression after
previous BP application in CNC-derived but not in mesoderm-
derived bone
Unilateral extraction of the mandibular first molar in animals
without previous BP application resulted in an increase in Cx43
protein expression over time, but not significantly when compared
with tibia bone samples that underwent osteotomy (tibia bone G3
vs jawbone G3 at week 12, p ¼ 0.154 and at week 16, p ¼ 0.484).
Interestingly, median Cx43 expression in jawbone was even lower
compared with tibial bone after 12 weeks, but increased substan-
tially and was comparable to tibial bone up to week 16. Tibial Cx43
expression did not show substantially changing expression levels
(p ¼ 0.469) at the two investigated timepoints (Fig. 4a). In BP-
treated animals, surgical intervention induced a significant
decrease in detected Cx43 in the CNC-derived jaw 16 weeks after
intervention compared with the concurrently treated mesodermal
bone (tibia G4 vs jawbone G4 at week 12: p < 0.01). Interestingly,
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Journal of Cranio-Maxillo-Facial Surgery 49 (2021) 738e747
this effect did not occur until postoperative week 12, and developed
during the following month. Regarding expression rates in the tibia,
BPs further induced Cx43 synthesis over the study period; finally,
expression resulted in even higher rates compared with non-BP-
treated animals (p ¼ 0.033) (Figs. 4b and 5; Table 1).
Focusing on the intervention-related differences in the jaw, BP-
treated animals showed significantly higher Cx43 expression at
postoperative week 12 (jawbone G3 vs jawbone G4 at week 12:
p ¼ 0.026). However, in the drug-treatment group, protein
expression decreased over the following month so that BP-treated
jaws presented significantly lower median values at the end of the
investigational period (jawbone G3 vs jawbone G4 at week 16:
p ¼ 0.005) (Fig. 6a; Table 1). In contrast, surgical intervention alone
only moderately increased Cx43 expression in the investigated
tibial bone samples (tibia G3 vs tibia G4 at week 12: p ¼ 0.469),
whereas previous BP treatment induced a significant increase in
protein expression as detected 16 weeks postoperatively (tibia G3
vs tibia G4 at week 16: p ¼ 0.033) (Fig. 6b; Table 1).
4. Discussion
Antiresorptive agents are known to maintain bone strength via
(1) halting the osteoclast-induced loss of bone mineral content and
(2) their anti-apoptotic effects on osteoblasts and osteocytes, thus
affecting both osteoblastic and the osteoclastic aspects (Bellido and
Plotkin 2011). Aside from its role as gap junction and hemichannel,
Cx43 has been shown to interact with intracellular structures and
signaling molecules through a cytoplasmic C-terminal tail (Plotkin
and Bellido 2013). In osteoblasts and osteocytes bisphosphonates
trigger the Src/ERK-pathway, promoting p90ERK phosphorylation
and finally protecting these cells from apoptosis. Some authors
even claim that Cx43 is required for the bisphosphonate-induced
effects on bone tissue, and regulates the maintenance of osteo-
cyte viability (Lezcano et al., 2012). In cell cultures, bisphosphonate
has shown an additive effect on Cx43 protein expression in a dose-
dependent manner within 24h of application (Jeong et al., 2013). In
contrast, it has also been shown that zoledronic acid decreases
Cx43 expression and therefore leads to the deterioration of the
R.H.M. Preidl, K. Amann, M. Weber et al. Journal of Cranio-Maxillo-Facial Surgery 49 (2021) 738e747

Fig. 3. Immunohistochemical Cx43 staining showed expression in mesoderm-derived tibial bone and cranial neural crest (CNC)-derived jawbone 12 weeks postoperatively in the
respective groups: a) and b) Group 1 (G1, untreated controls); c) and d) Group 2 (G2, BP application for 8 weeks); e) and f) Group 3 (G3, after molar extraction in the jaw and
osteotomy with osteosynthesis in the tibia); g) and h) Group 4 (G4, BP treatment prior to molar extraction in the jaw and osteotomy with osteosynthesis in the tibia). Sections are
presented at a magnifications of 5 and 200  , respectively.

osteocytic-canalicular system in bone (Cui et al., 2018). In our
analysis, we could not find significant changes in Cx43 expression
in zoledronate-treated animals weeks after treatment with regard
to the jaw or tibia. However, the CNC-derived jawbone presented
significantly higher Cx43 expression levels compared with tibial
bone 16 weeks after cessation of BP treatment.
We already know that a loss of Cx43 in the context of fracture
healing is associated with decreased bone formation and even bone
resorption, leading to lower mineral density and reduced cortical
thickness (Loiselle et al., 2013). Cx43 expression usually rises
initially after fracture, with detectable mRNA decreases over the
subsequent weeks depending on the animals’ age (Meyer et al.,
2006). In adult rats, aged around 26 weeks, Cx43 content in oste-
ocytes was shown to have decreased non-significantly 6 weeks
after femur fracture compared with previous time points. With
regard to our analysis in adult animals aged between 24 and 32
weeks, we were able to detect unaltered high levels of Cx43 in the
tibia even 16 weeks after the fracture event, which is in accordance
with the data in the literature relating to older animals aged around
50 weeks (Meyer et al., 2006). Interestingly, only in the jaw did
Cx43 expression after tooth extraction show further increases at
the second time point of the investigation compared to the previ-
ous time point, and even exceeded expression levels in the tibial
bone; however, this difference was non-significant. This result
might indicate the specific regulatory capability of CNC-derived
bone compared with mesoderm-derived bone. Furthermore, one
has to consider that aging is a factor that leads to reduced Cx34
expression and subsequent reduction in bone turnover over time,
especially in non-mechanically loaded bone (Davis et al., 2018). This
might explain the decrease in Cx43 expression in the non-treated
control group in our analysis. However, expression was still
significantly higher compared with tibia bone.
Another difference between CNC- and mesoderm-derived bone
relates to osteocyte and matrix composition (Leucht et al., 2008a,b).
From a mechanical point of view, cranial bone strain levels are
lower compared with the rest of the skeleton. In addition, although

742
R.H.M. Preidl, K. Amann, M. Weber et al.
Fig. 4. a) After extraction of a mandibular molar and osteotomy in the jaw Cx43 expression did not differ significantly between CNC- and mesoderm-derived tissue. Surgical
interventions with a history of a BP pretreatment for 8 weeks caused a significant decrease in Cx43 expression in the jaw, whereas tibial bone presented further increases in protein
expression.
not being exposed to a continuously weight-bearing mechanical
component to stimulate bone remodeling, CNC-derived bone is
resistant to postmenopausal hormonal changes and to
glucocorticoid-induced osteoporosis (Rawlinson et al., 2009).
Genetically, numerous genes and transcriptional factors are pre-
dominantly expressed either in bone originating from the CNC or in
mesodermal limb bone. In rats, for example, Dlx5 and Twist1 are
more highly expressed in CNC-derived bone compared with non-
cranial bone. Additionally, some genes, like Msx2 and Runx1, are
known to be selectively expressed in facial bone in rats and not in
the limbs, suggesting a ‘positional identity’, with distinctive
expression patterns in different bones throughout the skeleton
(Rawlinson et al., 2009). Furthermore, the receptor activator for
nuclear factor kB ligand (RANKL) is highly expressed in CNC-
derived jawbone, regulating bone turnover, tooth eruption, and
bone growth (Ealba et al., 2015; Uribe et al., 2018).
During bone healing, a unique stem cell population d
depending on the bone’s lineage d is present for regenerating bony
defects, indicating that skeletal stem cells also possess a ‘positional
memory’ even after transplantation. Moreover, skeletal progenitor
cells from the two lineages d mesoderm- and CNC-derived d
present different osteogenic potentials and proliferative capacities
(Leucht et al., 2008a,b). In chicks, Cx43 deficiency causes reduced
mandibular bone formation during embryological development,
potentially associated with a downregulation of Msx-1 (Batra et al.,
2012). Molecular signaling that regulates bone formation and
repair, as performed by the Wnt-signaling pathway, is present
during bone healing independently of the tissue’s embryological
lineage (Leucht et al., 2008a,b).
Via a selectively higher expression rate of Cx43 in the jawbone
compared with mesoderm-derived bone, BP effects on bone cells
might predominantly occur in CNC-derived bone tissue and
therefore cause MRONJ selectively in the jaw. In our study, surgical
intervention initially decreased gap junction expression in jawbone
compared with tibial bone, but this expression then increased
substantially in the jaw with ongoing healing progression. The
specific regulation of jawbone protein expression was also
observed as previous bisphosphonate application led to a signifi-
cant decrease in Cx43 in the CNC-derived jawbone. We could show,
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Journal of Cranio-Maxillo-Facial Surgery 49 (2021) 738e747
for the first time, that this effect persists, in contrast to the signif-
icantly increased Cx43 expression in mesoderm-derived bone in
the same organism 16 weeks postoperatively. Even though Cx43
expression was higher 12 weeks after operation in the BP-treated
jawbone compared with the non-treated jawbone, this expres-
sion pattern switched within the healing progression so that the
osteocytes in BP-affected bone finally presented significantly fewer
Cx43 gap junctions and hemichannels. The subsequent decrease in
localized healing capacities in the jawbone might explain the
occurrence of wound-healing disorders and finally the develop-
ment of osteonecrosis in the jaw. This hypothesis is supported by
data showing selective suppression of the RANKL/OPG-ratio and
Wnt-expression in BP-treated jaw in contrast to injury sites in
peripheral bone (Gong et al., 2017). Indicating a predominant
decrease in bone turnover and remodeling in the jaw, which has
already been demonstrated in human tissue samples (Wehrhan
et al., 2011), these aspects, together with the results presented in
our analysis, are important etiological factors in explaining the
development of BRONJ. At this stage, we hypothesize that MRONJ
caused by treatment with anti-RANKL humanized antibodies, like
denosumab, might explain its site-specific occurrence because of a
predominant RANKL suppression in CNC-derived bone. Until now,
it has been unclear whether there are also site-specific effects
concerning anti-angiogenic drugs, such as VEGF inhibitors. Since
this group of medications is also known to cause wound healing
disorders and even osteonecrosis in the jaw, one might speculate
that the effects of these drugs might be also be influenced by a site-
specific protein expression pattern in endothelial cells within the
jawbone area. Nevertheless, there still is a debate on the effects of
the microbial flora of the oral cavity on initiation or at least pro-
gression of MRONJ in jawbone (Koth et al., 2016). In our analysis,
the microbial loading of the mandibular wounds was not examined.
Previous studies investigating drug-associated bony changes in
mandible and femoral bone in rats, without any surgical inter-
vention or bone alteration, have revealed an increased incidence of
localized mandibular inflammation, resulting in osteonecrosis in
two out of ten animals (Senel et al., 2010). These BP-treated animals
did not present any sign of inflammation in the skeletal bone, and
the authors state that no Actinomyces colonies were observed in
R.H.M. Preidl, K. Amann, M. Weber et al. Journal of Cranio-Maxillo-Facial Surgery 49 (2021) 738e747

Fig. 5. Immunohistochemical staining for Cx43 in mesoderm-derived tibial bone and CNC-derived jawbone 16 weeks postoperatively. Groups are categorised as follows: a) and b)
Group 1 (G1, untreated controls); c) and d) Group 2 (G2, BP application for 8 weeks); e) and f) Group 3 (G3, after molar extraction in the jaw and osteotomy with osteosynthesis in
the tibia); g) and h) Group 4 (G4, BP treatment prior to molar extraction in the jaw and osteotomy with osteosynthesis in the tibia). Sections are presented at a magnifications of 5
and 200  , respectively.

these animals at the respective sites. Consequently, some authors
state that localized bony infection can be seen in the context of a
secondary infection aggravating a pre-existing inflammation
(Wang et al., 2019). However, we were able to show that Cx43
expression in jawbone with surgical intervention was significantly
higher compared with jawbone that underwent the same operative
procedure, presumably with a comparable loading of microbial oral
flora, but with BP exposure before the intervention (Fig. 6a).
Therefore, these results might indicate a less important role of the
microbial oral flora during MRONJ initiation, which needs to be
investigated in further experiments, and is a limitation of this
study.
In addition to the reported patient series relating to BRONJ
selectively affecting the lower or upper jaw, an increasing number
of published case series presenting BP-related osteonecrosis of the
inner ear have emerged (McCadden et al., 2018). From an embry-
ological perspective, the middle and outer ears originate from the
first two branchial arches and are characterized by a distinctive
Msx1 expression pattern (Satokata and Maas 1994). At this stage,
we speculate that osteonecrosis of the middle ear might be based
on the embryological lineage of the bony tissue and its site-specific
protein expression. Interestingly, MRONJ selectively affecting the
mandibular condyle and the joint has not been reported in the
literature so far. As neural crest-derived cells contribute to the
development of the ascending aorta and the aortic valve in early
embryological stages, bisphosphonates might also selectively affect
these cells and thereby influence cardiovascular disease (Peterson
et al., 2018). In rabbits, pamidronate concentration 24 h after
application was significantly higher in the aortic valvular ring in
atherosclerotic tissue samples compared with healthy controls
(Ylitalo et al., 1996).
Over the last decade, numerous animal models investigating
histopathological and therapeutical aspects in the context of BRONJ
have been published in the literature (Table 2) (Howie et al., 2015).
Focusing on the applied dosage and duration of BPs, the presented
studies vary substantially regarding these factors; however,
osteonecrosis was found in all of the listed analyses. Nevertheless,
the investigational timepoints also differed tremendously among

744
R.H.M. Preidl, K. Amann, M. Weber et al. Journal of Cranio-Maxillo-Facial Surgery 49 (2021) 738e747

Fig. 6. a) In selective comparisons of Cx43 expression in surgically treated jawbone, previous BP treatment significantly decreased Cx43 expression in the long term, whereas
expression in non-BP-affected bone increased further. b) In contrast, during regeneration in mesoderm-derived bone tissue, Cx43 expression was significantly higher in tibial bone
of BP-treated animals compared with non-BP-treated animals.

Table 2
Summary of published BRONJ rat models over the previous 3 years. Applied bisphosphonate doses are unaltered compared with previous models presented by Howie et al.
(Howie et al., 2015) in 2015 (Kim et al., 2015; Silva et al., 2015; Takaoka et al., 2015; Silveira et al., 2016; Zandi et al., 2016; Oliveira et al., 2017; Vidal-Gutierrez et al., 2017;
Koneski et al., 2018; Kun-Darbois et al., 2018; Wang et al., 2019).

Publication Investigated bone Species Applied BP Dosage per weekly injection [mg/kg bodyweight] Duration of BP treatment

Wang J.Y. et al., 2019 Mandible and maxilla Rat Zoledronate 0.066 4 weeks
Koneski F. et al., 2018 Mandible Rat Zoledronate 0.06 5 weeks
Kun-Darbois J.D. et al., 2018 Mandible Rat Zoledronate 0.10 10 weeks
Oliveira C.C. et al., 2017 Mandible Rat Zoledronate 0.20 8 weeks
Vidal-Gutie
rrez X. et al., 2017 Maxilla Rat Zoledronate 0.06 2 weeks
Silveira F.M. et al., 2016 Maxilla Rat Zoledronate 0.60 12 weeks
Zandi M. et al., 2016 Mandible Rat Zoledronate 0.06 5 weeks
Kim J.W. et al., 2015 Mandible Rat Zoledronate 0.10 14 weeks
Silva P.G. et al., 2015 Mandible Rat Zoledronate 0.20 3 weeks
Takaoka K. et al., 2015 Maxilla Rat Zoledronate 0.035 2 weeks

the listed analyses, ranging from 2 to 14 weeks after surgical
intervention. For our experiments we decided to sacrifice the ani-
mals 12 weeks postoperatively in order to clearly observe the
generated pathological environment in the affected bones. How-
ever, since bone healing and remodeling were still ongoing at this
time, we added an additional time of assessment 4 weeks later to
exclude long-term changes. The animal model used in this exper-
iment to investigate Cx43 expression has already been used by
other groups in the context of MRONJ research (Agaçayak et al.,
2014; Jabbour et al., 2014; Howie et al., 2015; Wang et al., 2019).
Using comparable BP dosage schemes and time intervals, other
studies also describe only few cases developing fulminant clinical
symptoms of MRONJ within the respective treatment groups after
tooth extraction. However, delayed bone remodeling and wound
closure in the jaw within BP-treatment groups were recognized and
reported by most authors (Williams et al., 2014; Wang et al., 2019).
Histological sections revealed a significantly higher ratio of empty
lacunae to viable lacunae in the respective bone areas, and in some
cases even sequestra were observed. Additionally, TRAP expression
in osteoclasts as well as ALP within the affected jaw area were both
significantly reduced in BP-treated animals (Williams et al., 2014).
745
It has been reported that the rate of induced ONJ lesions in small
animal models varies among studies, having been observed in
around 15e50% of the animals (Williams et al., 2014). This clearly
shows the capability of the applied model. With regard to our
investigational groups, we could observe enoral wound de-
hiscences within the alveolar region in BP-treated animals too; in
four out of ten animals with tooth extraction in combination with
prior BP application we could observe wound disorders with
partially exposed alveolar bone.
5. Conclusion
We were able to show that Cx43 is expressed and regulated in a
site-specific manner regarding neural crest- and mesoderm-
derived bone tissue. In accordance with findings in the current
literature, the specific occurrence of BP-related osteonecrosis of the
jaw might be related to the embryological origin of the jaw tissue
per se. Consequently, life-long, site-specific gene and protein
expression is determined, potentially causing a susceptibility to BPs
in neural crest-derived tissue.
R.H.M. Preidl, K. Amann, M. Weber et al.
Author contributions
FW designed the study; RHMP, MW, CG, MS, MR, and FW
collected the data; RHMP, FW, KA, MK, JR, and FN drafted the
article; all authors approved the final version.
Declaration of Competing Interest
The authors of this manuscript have no conflicts of interest to
disclose.
Acknowledgments
This study was supported by the Deutsche For-
schungsgemeinschaft (DFG) (No. WE5273/1).
We thank Susanne Schoenherr and Elke Diebel for their support
during the immunohistochemical staining.
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