Bone 143 (2021) 115765

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Bone
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Review Article

A review of the biomarkers and in vivo models for the diagnosis and
treatment of heterotopic ossification following blast and
trauma-induced injuries
Zepur Kazezian *, Anthony M.J. Bull
Centre for Blast Injury Studies, Department of Bioengineering, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom

A R T I C L E I N F O A B S T R A C T

Keywords: Heterotopic ossification (HO) is the process of de novo bone formation in non-osseous tissues. HO can occur
Heterotopic ossification (HO) following trauma and burns and over 60% of military personnel with blast-associated amputations develop HO.
Ectopic bone This rate is far higher than in other trauma-induced HO development. This suggests that the blast effect itself is a
Biomarkers of HO
major contributing factor, but the pathway triggering HO following blast injury specifically is not yet fully
Signalling pathways of HO
identified. Also, because of the difficulty of studying the disease using clinical data, the only sources remain the
in vivo models of HO
relevant in vivo models. The aim of this paper is first to review the key biomarkers and signalling pathways
identified in trauma and blast induced HO in order to summarize the molecular mechanisms underlying HO
development, and second to review the blast injury in vivo models developed.
The literature derived from trauma-induced HO suggests that inflammatory cytokines play a key role directing
different progenitor cells to transform into an osteogenic class contributing to the development of the disease.
This highlights the importance of identifying the downstream biomarkers under specific signalling pathways
which might trigger similar stimuli in blast to those of trauma induced formation of ectopic bone in the tissues
surrounding the site of the injury. The lack of information in the literature regarding the exact biomarkers
leading to blast associated HO is hampering the design of specific therapeutics. The majority of existing blast
injury in vivo models do not fully replicate the combat scenario in terms of blast, fracture and amputation; these
three usually happen in one insult. Hence, this paper highlights the need to replicate the full effect of the blast in
preclinical models to better understand the mechanism of blast induced HO development and to enable the
design of a specific therapeutic to supress the formation of ectopic bone.

1. Introduction
Heterotopic ossification (HO), which is the process of extra-skeletal
bone formation in the non-osseous soft tissues [1], was first described
in the late 19th century by Riedel [2], and then in the early 20th century
by Dejerne and Ceillier who described HO in soldiers following spinal
cord injuries in World War I [3,4]. The term “Heterotopic ossification”
which indicates the bone formation in another place comes from the
Greek heteros topos, which means different place, and the Latin ossifi­
catio, which means bone formation [5].
HO can be hereditary which is classified into fibrodysplasia ossifi­
cans progressiva (FOP) [6] and progressive osseous hyperplasia (POH)
[7,8], and non-hereditary which develops as a result of injury,
arthropathy, and age. While hereditary HO is rare, non-hereditary HO is
acquired and includes traumatic and neurogenic HO that might occur
due to several factors including soft tissue injury, trauma or surgery,
central nervous system and spinal cord injury, burns, and amputations
[9–14]. There is a 10–20% risk of HO associated with brain and spinal
cord injury [15,16], yet a much higher percentage of around 63–64%
with blast-induced or other combat-related amputations develop ectopic
bone [17–20]. Of these combat-related amputations, 11% require
further revision [19]. HO can cause pain, neurovascular entrapment,
reduced range of motion, and, in the amputees, difficulty in wearing
prostheses due to frequent wound breakdown. The risk factors for HO in
military amputees include age, blast, site of amputation, injury severity,
and increased number of debridements prior to wound closure [21].
While prophylactic treatments such as non-steroidal anti-inflamma­
tory drugs (NSAID)s and radiation therapy are used to treat HO, the only

* Corresponding author at: Imperial College London, Bessemer Building, South Kensington Campus, London SW7 2AZ, United Kingdom.
E-mail address: z.kazezian@ic.ac.uk (Z. Kazezian).

https://doi.org/10.1016/j.bone.2020.115765
Received 7 May 2020; Received in revised form 29 October 2020; Accepted 18 November 2020
Available online 4 December 2020
8756-3282/© 2020 Elsevier Inc. All rights reserved.
Z. Kazezian and A.M.J. Bull
effective intervention for HO resulting from complex blast injuries re­
mains surgical excision of the ectopic bone which has multiple draw­
backs and risks [22], bearing in mind that even this treatment cannot be
applied to some patients due to the nature and the location of the formed
bone [1]. Moreover, despite the excision of the ectopically formed bone,
HO will recur following the surgery in up to 27% of patients who have
partial excision and 7% of patients who have complete excisions [22].
Due to the high prevalence of HO following blast injuries, and,
because the mechanisms of HO initiation and progression following
blast are not yet fully understood, the aim of this paper is to conduct a
review of the key biomarkers and signalling pathways identified which
trigger the development of the ectopic bone in trauma-induced and
specifically blast associated HO as well as to summarize the most up to
date blast injury associated HO in vivo models. This will enable the
elucidation of the molecular mechanisms that can trigger HO and thus
provide new opportunities for researchers to design relevant preclinical
models to investigate the mechanisms and design and test novel thera­
pies for HO following blast.
2. Heterotopic ossification development, classification and
detection
Survival of wounded military personnel has increased over recent
years to up to 88% in the wars in Iraq [23]. 70% of the injuries of these
survivors were musculoskeletal with the extremities being the most
injured which has led to a high percentage of survivable blast trauma
related amputations [24–26]. Blast injuries are characterized as pri­
mary, secondary, tertiary, quaternary or quinary [27]. Primary blast
injuries are caused by the direct effect of the blast overpressure on tis­
sues, secondary blast injuries are caused by objects or fragments pushed
by the force of the blast wave, tertiary blast injuries are due to whole
body displacement by the explosive energy of the blast and quaternary
blast injuries are caused by other mechanisms that can cause harm, such
as burns. Recently, blast injuries have been also characterized as quinary
which refer to delayed effects from blast exposure, such as chronic pain,
immunosuppression or malnutrition [28,29]. Blast-induced traumatic
amputations can occur due to a combination of primary, secondary, and
tertiary mechanisms [30–32] and the majority of these present HO [20].
Certain conditions need to be present for HO to occur, such as
osteogenic progenitor cells, appropriate environment and a triggering
incident [33]. One of the most important factors which is considered to
play a crucial role in HO development is the set of complex inflamma­
tory reactions with diverse cross-talking inflammatory stimuli and sig­
nalling cascades [34]. These multiplicity of inflammatory cascades
means that there is controversy in what is the exact trigger that leads to
HO initiation and progression and therefore there is a need to focus on
the early stages of the disease. Early clinical detection of ectopic bone
formation is difficult as there is currently no absolutely reliable radio­
graphic method that can confirm HO until there is calcified bone
present.
Multiple classifications of blast injury associated ectopic bone are in
use; a morphological approach by Evriviades et al has two types: Type 1
includes spike like bone, growing into the surrounding muscles, which is
continuous with the terminal site of the amputated bone; and Type 2 in
which bone grows in sheets into the surrounding muscle assuming the
shape of beetle’s exoskeleton not necessarily from the amputated bone
end [35]. The quantitative approach proposed by Potter et al uses ra­
diographs of the remaining limbs of amputees to classify ectopic bone
due to HO into: 1) mild HO covering <25%, 2) moderate HO expanding
25–50%, and 3) severe HO dominating >50% of the cross-sectional area
of the remaining limb on any of the antero- posterior or lateral radio­
graphs [14].
Imaging ectopic bone is challenging and can be conducted using 2D
X-rays, CT and Micro-CT, ultrasound, Positron Emission Tomography
(PET), and Magnetic Resonance Imaging (MRI). The most common
method for mature ectopic bone detection is X-ray because it is
2
Bone 143 (2021) 115765
affordable and quick [36,37]. CT can be used similarly to give a three-
dimensional representation of mineralised tissues. Micro-CT has been
also utilised for HO detection in studies involving rodents [38] that were
able to show ectopic bone in a rodent model of blast HO as early as 3
weeks post insult [39]. Ultrasound can be used for HO detection in cases
of spinal cord injuries [40]. This low-cost and safe method provides real-
time evidence of the variations in stiffness of soft tissues before radio­
graphic detection, yet it is highly operator dependent [41]. MRI due to a
lack of ionising radiation has great potential, but it cannot distinguish
adequately between immature ectopic bone and other pathologies;
when there is doubt regarding such results, CT scan follow ups are
required. Unlike MRI, CT scanning is better at identifying mature ectopic
bone than immature bone as its signal intensity is similar to that of
normal bone [42]. Other imaging modalities such as PET utilising ra­
diotracers can be used in combination with CT and offers potential as a
biomarker of early HO development [43].
Thus far, the earliest detection of HO achieved in an in vivo model is
by Near-Infrared (NIR) optical imaging [44]. In this Achilles tenotomy/
burn model of HO, the ectopic bone was detected as soon as five days
post trauma with non-invasive NIR imaging. Validation was done in
parallel with confocal microscopy. Furthermore, Raman spectroscopy
[45,46] has been used for the visualization of HO in a mouse model of
burn-induced HO [47]. This could potentially detect ectopic bone as
early as NIR.
In summary, X-rays are easy, yet detect HO quite late (after miner­
alisation) and don’t quantify the 3D shape and severity; MRI similarly
detects only mature ectopic bone; ultrasound produces variable results
due to a high inter-operator variability; CT (and micro CT) increases the
ionising radiation, yet addresses the planar issue and so enables quan­
tification of the severity of HO which is helpful in clinical decision-
making [48]; NIR imaging detects HO earlier than CT, yet has low res­
olution; and PET/CT shows some early promise as a biomarker of early
HO.
2.1. Key molecular markers leading to heterotopic ossification
To date, the cellular and molecular markers that lead to HO and thus
ectopic bone formation are not yet fully known. Similarly, therapeutic
options are limited and non-specific, and include NSAIDs, radiation
therapy or surgical excision. Furthermore, as described above, there are
no specific diagnostic tools to detect early HO in the clinic. Hence, the
identification of biomarkers of the early stages of HO should be the focus
of research. This will enable future work to be directed towards the
diagnostics and design of therapeutic interventions.
For HO to occur, stem cell recruitment, proliferation, and differen­
tiation into osteogenic cells need to be initiated. In vitro studies repre­
senting the mitogenic and osteogenic effects of serum from patients with
trauma to the central nervous system on osteoblastic, mesenchymal and
fibroblastic cells give credence to the association with humoral factors
[49–54]. The good blood supply to the skeletal muscles enables stem
cells to acquire osteogenic differentiation capabilities [55], thus
providing evidence for the increased incidence of HO in the skeletal
muscles compared to other soft tissues [56–58]. Studies on adult human
skeletal muscle cells that differentiated into osteoblastic cells in vitro
formed new bone when re-implanted into animals [59–61]. Studies have
shown that the key trigger to initiate HO might be local inflammation
following trauma, including spinal cord injury, traumatic brain injury,
surgical intervention, blast and deep burns [62–64]. Inflammatory cells,
such as macrophages, lymphocytes, and mast cells surrounding the
perivascular area of the newly formed ectopic bone as a result of damage
to skeletal muscle cells and tissue hypoxia can prompt the proliferation
and differentiation of progenitor cells. Yet, significant clinical and
experimental evidence indicates that the bone morphogenetic protein
(BMP) family plays a fundamental role in the development of acquired
HO. It is well known that the BMP family, which is part of the TGF-β
superfamily, has a crucial role in chondrogenesis and osteogenesis
Z. Kazezian and A.M.J. Bull Bone 143 (2021) 115765

through recruitment of mesenchymal cells and stimulating their differ­
entiation into chondrocytes and osteoblasts (Figs. 1 and 2) [65]. BMPs,
including BMP2, BMP4, BMP7 and BMP9 have been tested previously in
the context of HO by injecting into the muscle tissue of mice [66,67].
They have also been found to be over-expressed in human ectopic bone
[68,69] which might indicate that they might play a key role in HO
(Fig. 1). Unlike other BMPs, BMP2 is needed for MSC recruitment and
differentiation as well as appropriate fracture healing; consequently,
experiments with knock out mice showed that a lack of BMP2 leads to
failure in fracture healing [70]. Many other biomarkers have been
identified as being correlated with ectopic bone development after blast
and other trauma, these are reported in Fig. 1 and Table 1.
3. Signalling pathways identified in acquired HO
Currently, there is lack of specific biomarkers and signalling path­
ways that can identify HO associated with blast injuries. However,
biomarkers and signalling pathways identified in other traumatic and
genetic types of HOs might help in understanding the mechanisms of HO
following blast.
Acquired HO is a consequence of traumatic tissue damage. It is also a
main clinical challenge because of the unclear primary mechanisms
underlying the HO initiation and progression which are likely to be far
more variable than hereditary HO. In vivo models would be suited to
assess these mechanisms, yet there are only few suitable in vivo models.
The research to date has found that BMP signalling plays a key role in
acquired HO. Specifically, increased levels of BMP2 are associated with
acquired HO [81]. In a traumatic injury model, expression of various
osteogenic gene transcripts, including BMP-2, BMP-3, and SMAD1
downstream of the BMP signalling pathway was found elevated in
wound tissue. Also, the expression of a type I BMP receptor a (BMPRIa),
was increased [71]. Type I BMP receptor (BMPRIa) and type II BMP
receptor (BMPRII) were also up-regulated seven days post SCI, and
phosphorylation of SMAD and translocation to nuclei was observed
[82]. An in vitro study showed that low amounts of the fibroblast growth
factor 2 (FGF2) seemed to increase BMP2-induced ectopic bone forma­
tion via increased BMPRIb expression and SMAD1 phosphorylation
while high dosages of FGF2 hindered MSC differentiation into osteo­
genic cells [83]. Furthermore, TGF-β2 was increased threefold in
immature compared to mature ectopic bone rendering it a crucial
component of the process of HO after THA [84]. TGF-β uses different
intracellular signalling pathways other than SMAD signalling to control
a wide range of cellular functions. Quick activation of Ras and or
extracellular receptor kinase (ERK1/2) by BMP is also reported in
myoblasts, osteoblasts, and stem cells [85,86]. ERK signalling was also
detected in an in vitro MC-3 T3 and in vivo mouse HO model induced by
connexin 43 (Cx43). ERK signalling induced by Cx43 had an essential
role in HO development, because inhibition of Cx43 and ERK down-
regulated the expression of Runx2, bone sialoprotein (BSP) and
collagen 1 (Col-1) both in vivo and in vitro. Therefore, low levels of Cx43
and ERK lowered the risk of HO recurrence [87]. Mitogen-activated
protein kinase (MAPK) signalling was also found upregulated when
human adipose stem cells (hASC) treated with trauma HO+ patient’s
serum vs HO− serum. Genomic analysis showed that hASCs treated with
serum from individuals who developed HO had affected the expression
of the activator protein 1 (AP1) signalling. Additionally, significant
downregulation in FOS gene expression in hASCs treated with serum
from individuals with HO was detected. Furthermore, the MAPK sig­
nalling pathway as well as downstream genes associated with the MAPK
were upregulated in hASCs following serum exposure from individuals
with HO [88].
These signalling pathways have further crosstalk with each other to
complete their role in the disease mechanism. BMP signalling is sug­
gested to crosstalk with other key regulators of HO which are Hif1α and
mammalian target of rapamycin (mTOR) pathways in the context of HO
[89]. In vitro long-term hypoxic environment (3%) exposed osteoblasts
shows up-regulated BMP2 through the stimulation of the mTOR and
HIF1-α signalling pathways (Fig. 2). Hypoxia increases mTOR phos­
phorylation and stabilises the activity of the mTOR and HIF1-α signal­
ling pathway molecules. On the other hand, the inhibition of mTOR and
HIF1-α signalling impairs the hypoxia induced BMP2 expression [90]
(Fig. 2). Another example is the crosstalk between the BMP signalling
and Wnt B-catenin as well as the ERK signalling pathways through the
BMP downstream mediator RUNX-2 [91]. There is still controversy
around BMP and Wnt functions in osteogenesis, suggesting that Wnt is
the major controller of osteogenesis in BMMSCs while BMP signalling is
the major controller of osteogenesis in adipose derived mesenchymal
stem cells (ADMSCs) [92]. Overall, most studies suggest that the impact
of Wnt signalling is less important than BMP signalling when it comes to
MSCs differentiation into osteogenic cells. Fig. 2 summarises the

Fig. 1. Biomarkers identified in preclinical and clinical studies after HO development as a result of blast injuries. These data are summarised in Table 1. Magnifying
the amputated bone through the fracture resulted from blast injury, there are inflammatory cells including lymphocytes and macrophages which play a crucial role in
releasing cytokines/biomarkers such as BMPs, TGF-β and SMADs which can recruit stem cells such as bone marrow mesenchymal stem cells (BMMSC) and induce
their differentiation into chondrocytes and osteoblasts resulting in the ectopic bone formation. On the left, there are three groups of biomarkers summarised from
studies in humans (top), in vivo rat models (middle) and common in both humans and rats (bottom).

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Z. Kazezian and A.M.J. Bull Bone 143 (2021) 115765

Fig. 2. The canonical and non-canonical pathways of BMP/TGF-β family, mTOR and Wnt signalling pathways that can lead to heterotopic ossification. The canonical
pathway is mediated via different SMADs (SMAD1/5/8 and SMAD2/3) that can be phosphorylated (activated) then translocate to the nucleus and act as activators (R-
SMAD) or inhibitors (SMAD6/7) to the transcription factors of osteogenic or chondrogenic markers participating in bone formation. Non-canonical pathway can be
mediated via non-SMAD dependant pathways such as mitogen-activated protein kinase (MAPK) pathway activating downstream extracellular signal-regulated ki­
nases (ERK) and mitogen-activated protein kinase p38 (P38) which in turn will activate transcription factors leading to the transcription of chondrogenic or oste­
ogenic markers. There might be also a crosstalk between hypoxia mediated mTOR, HIF1-α and BMP pathways which can be inhibited by Rapamycin. Moreover, BMP
signalling might crosstalk with Wnt/B-catenin and the ERK signalling pathways through a downstream mediator such as RUNX-2.

pathways through which BMPs and the TGF-β family can induce chon­
drogenic and osteogenic markers in an HO environment through cross
talk with other signalling pathways.
3.1. Is there an ultimate treatment for heterotopic ossification following
blast injury?
The current therapeutic interventions for HO treatment at early
stages include NSAIDs, bisphosphonates, and radiation therapy [93,94].
Different forms of NSAIDs are recommended for prophylaxis such as
indomethacin, or aspirin-like medications which inhibit non-specifically
the cyclooxygenase enzyme which is vital in both endochondral bone
formation and HO development [95]. NSAIDs are widely used in the
treatment of the inflammation stimulated in the muscles and the soft
tissue which might lead to HO following THA [96], traumatic brain
injury and spinal cord injury [97]; their mode of action is through
blocking the enzymes that trigger inflammatory reactions.
NSAIDs show higher efficacy when they are administered at the
earlier phases rather than later phases of HO. Radiation therapy is also
used for treating HO, but it needs to be administered within the first 48 h
of injury [5]. The potential risks associated with radiation therapy is
malignancy, genetic mutations, impaired fracture healing and wound
complication while NSAIDs are also correlated with gastrointestinal
irritation, renal toxicity, platelet deficiency and increased bleeding [98].
In advanced stages of HO, the only successful intervention is surgical
excision of the ectopic bone which might be performed more than once.
In such cases imaging is useful to assess the HO severity prior to the
surgery. The timing for the bone excision surgery is crucial, because, if
the ectopic bone is excised too early, some complications such as hae­
morrhage and postoperative recurrence of HO are likely. If the ectopic
bone is excised too late, serious complications such as joint ankylosis
may occur [36,37]. Although there is insufficient evidence available to
define the most effective time point for ectopic bone excision, some
studies suggest that excision of the bone needs to be done after 6 months
of HO development, and to avoid the recurrence of HO a complete
excision should be achieved [22].
In terms of effectiveness, radiotherapy is not significantly more
effective than NSAIDs in the prevention of HO and vice versa; however,
both are more effective when they are used in combination [99]. While
the combination of NSAIDs and radiation therapy is the only choice for
early stage HO prevention, more specific drugs targeting the SMAD-BMP
pathway signalling are being developed. The immunosuppressant
rapamycin may be a suitable target compound as it may interfere with
the HO at the level of two main signalling pathways through Hif1α [79]
and mTOR pathways [100] (Fig. 2). Despite this promising approach,
existing treatments for HO are insufficient for treating military ampu­
tees; the best therapeutic intervention is yet to be identified. The ideal
treatment would be the complete prevention of HO, and this can only be
achieved when all the pathophysiological causes leading to the ectopic
bone formation due to traumatic blast injuries have been identified.

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Z. Kazezian and A.M.J. Bull Bone 143 (2021) 115765

Table 1
Biomarkers of heterotopic ossification identified as a result of blast and other trauma.
Biomarker Model Cause Objectives Outcomes Ref

RUNX-2, OCN, NOG, SP-1, Rat Blast To identify early osteogenic markers that 1. Radiographs showed evidence of HO [39]
COL1α1, PHEX, and contribute to HO development development within 4 weeks
POU5F1 2. Starting day 3 and up to day 14 after injury,
chondrogenic markers such as collagen
type I alpha 1 (COL1α1), osteogenic marker
Runt-related transcription factor 2 (RUNX-
2), phosphate-regulating neutral endopep­
tidase, X-linked (PHEX) and POU domain
class 5 transcription factor (POU5F) and
proteins Noggin (NOG), osteoclacin (OCN)
and substance P-1 (SP-1) were upregulated
in the injured soft tissue compared to the
control or sham
3. Hypertrophic chondrocytes, cartilage, and
newly formed bone were detected in the
non-osseous tissues at the site of amputa­
tion by day 14
ALPL, BMP-2, BMP-3, Human Blast To determine the genomic profile of 1. At the initial debridement compared to the [71]
COL2A1, COLL10A1, osteogenic markers in blast related HO; control patients (no HO developed), the
COL11A1, COMP, CSF2, and to assess whether the mRNA wounds of personnel having HO developed,
CSF3, MMP8, MMP9, expression of the genes correlates with alkaline phosphatase (ALPL), BMP-2, BMP-
SMAD1 and VEGFA the protein expression and HO 3, collagen 2 alpha 1 (COL2A1), collagen
development 10 alpha 1 (COLL10A1), collagen 11 alpha
1 (COL11A1), cartilage oligomeric matrix
protein (COMP), colony stimulating factor
2 (CSF2), colony stimulating factor 2
(CSF3), matrix metalloproteinase-8
(MMP8), matrix metalloproteinase-9
(MMP9), SMAD1 and vascular endothelial
growth factor (VEGFA) were found upre­
gulated more than two folds
2. The same 13 genes were expressed at a
higher level in the final debridement
3. No significant changes between the initial
and the final debridement patients were
observed for the following genes ANXA5,
BGN, COL1A, COL3A1, COL5A1, COL12A1,
COL14A1, COL15A1, CTSK, ITGB1, MMP2,
SERPINH1 because they were highly
expressed in wounds of both cohorts
4. At the final debridement, there was a 2.5-
fold upregulation of BMP-2 in the wounds
of patients with HO development and there
was a high correlation between the tran­
script and the functional protein suggesting
that BMP-2 might serve as a biomarker for
war associated HO
IL-6, IL-10, MCP-1 (serum) Human Blast To identify the unique cytokines and 1. The rate of HO identified in the study [72]
IP-10 and MIP-1a (wound chemokines linked to HO after combat- population (24 patients) was 38%
Effluent) related trauma 2. An increased injury severity score was
correlated with the development of HO
3. Impaired healing and bacterial colonization
of wounds correlated with the development
of HO
4. Serum interleukin-6, interleukin-10, and
MCP-1 and wound effluent IP-10 and MIP-
1a were correlated with HO
ACTA, TNFα, BMP1, BMP3, Human Blast To identify the biomarkers of 1. OPN, RUNX2 and COL1A1 were [73]
TGF-β1, Fibronectin, and rat osteogenesis and fibrosis during the upregulated in rat muscle tissue at 7 days
SMAD3 and PAI-1 development of HO through comparing a and human muscle tissue at 10 days as a
rat blast model and a human muscle from result of traumatic blast injury
a blast injury 2. Based on the protein analysis, increased
expression of tissue fibrosis biomarkers
such as TGF-β1, Fibronectin, SMAD3 and
PAI-1 was found
3. Based on the gene analysis, biomarkers of
inflammation and fibrosis such as ACTA,
TNFα, BMP1 and BMP3 were found
upregulated in both rat and human samples
IGF1, OPN, MPO, RUNX2, Human Combination of different To identify biomarkers in HO+ patients 1. Insulin-like growth factor I (IGF1) was [74]
GDF2 or BMP-9 causes including brain injury, compared to HO− using systemic blood upregulated in HO+ samples
THA, traumatic surgeries, and tissue, and to compare the expression 2. Osteopontin (OPN), myeloperoxidase
burns with orthopaedic injuries of the identified biomarkers in tissue (MPO), runt-related transcription factor 2
and amputation (RUNX2), and growth differentiation factor
(continued on next page)

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Z. Kazezian and A.M.J. Bull Bone 143 (2021) 115765

Table 1 (continued )
Biomarker Model Cause Objectives Outcomes Ref

using semi quantitative and quantitative 2 or bone morphogenetic protein 9 (BMP-9)

protein analysis techniques were found downregulated in HO+ samples
OSC, OMD and COL1A2 Human Combination of different To identify biomarkers in HO+ patients 1. Osteocalcin (OSC) preprotein, [75]
causes including brain injury, compared to HO− after THA using osteomodulin and collagen alpha-1 (v)
THA, traumatic surgeries, systemic blood utilising iTRAK SRM-MS chain isoform 2 might serve as clinical
burns with orthopaedic injuries proteomic analysis biomarkers for HO detection
and amputation
CTX-I, PINP, OC Human THA To identify biomarkers associated to 1. Increase of >42% in osteoclast marker C- [76]
bone metabolism related to developing telopeptide of type-I collagen (CTX-I) at
HO after THA week 1 with a sensitivity of 89% and a
specificity of 82% at week 26 after surgery
2. Increase of >57% in osteoblast markers N-
terminal propeptide of type-I procollagen
(PINP) at week 6 with a sensitivity of 89%
and specificities of 82% in people devel­
oping HO
3. Increase of >13% in osteocalcin (OC) at
week 6 with a sensitivity of 56%, and
specificities of 91% in people developing
HO
Creatine Kinase Human Spinal cord injury To identify whether creatine kinase in 1. 14/18 patients who had normal levels of [77]
the serum of spinal cord injury patients creatine kinase, 13/18 didn’t have any
will serve as a marker of HO evidence of HO on the radiographs
2. 4/18 patients showed increased levels of
serum creatine kinase developed HO which
was evident through radiographs
α2-HS glycoprotein, CRP, Human Spinal cord injury To study the link between the α2-HS 1. Serum level of α2-HS glycoprotein was [78]
serum calcium, D-dimer, glycoprotein concentrations in the serum found significantly decreased in SCI
and BMP and the incidence of neurogenic patients with NHO as compared patients
heterotopic ossification (NHO) in without NHO
patients with spinal cord injury (SCI) 2. A significant increase of serum calcium, D-
dimer, BMP, and CRP was observed in SCI
patients with NHO compared to SCI
patients without NHO
Hif1α, SOX9 Mouse Trauma-induced (burn/ To identify whether targeting Hif1α with 1. Hif1α was upregulated in the three [79]
tenotomy), genetic, and a a drug treatment or conditional knockout different in vivo models of HO
hybrid model of genetic and can stop HO development 2. Pharmacological treatment targeting Hif1α
trauma-induced HO inhibits HO development as a result of
burn/tenotomy
3. Pharmacological treatment targeting Hif1α
inhibits HO resulted from hyperactivation
of ACVR1
4. Both pharmacological or genetic loss of
Hif1α in mesenchymal cells prevent their
accumulation and eventually HO formation
Hif1α and Runx2 Rat Achilles tenotomy To study the effect of inhibition of Hif1α 1. The inhibition of hypoxia-inducible factor [80]
and Runx2 in an animal model of HO 1-alpha (Hif1α) hindered osteoblast prolif­
induced by Achilles tenotomy eration but not differentiation
2. The inhibition of Runx2 didn’t impair the
proliferation of osteoblasts but only the
differentiation
3. The inhibition of both Runx2 and Hif1α by
siRNA treatment inhibited the osteoblast
differentiation
4. Inhibition of Hif1α prevented HO formation
only at the initial stage
5. Inhibition of Runx2 prevented HO
formation at the initial stage and after
chondrogenesis

4. In Vivo models used to study heterotopic ossification
Studying HO in humans is difficult due to obvious ethical issues in
accessing the clinical results and clinical issues associated with the
lifesaving treatment that is the focus at the initial stages of ectopic bone
formation. Therefore, blast injuries must be replicated in in vitro, ex vivo
and in vivo models to identify the disease initiation, development and
eventually specific treatment modalities. Existing in vivo blast induced
HO models require three essential characteristics: blast, fracture and
amputation with a single insult to adequately represent the insult
received in military blast injuries. In vivo studies developed in the last
decade have used various rat models because of its availability and low
cost. Some of these models used a shock tube to investigate the impact of
different magnitudes of the blast pressure on inflammation and its
duration in the animal [101], others used a pentaerythritol tetranitrate
explosive, either submerged beneath a water tank [102] or sand
container [103] to induce fracture. The blast associated amputations in
these models have contributed to our knowledge regarding HO forma­
tion following blast. For instance, one of these studies analysed omics
data and identified key biomarkers such as RUNX-2, OCN, NOG, SP-1,
COL1α1, PHEX, and POU5F1 [39] which have some biomarkers in
common with trauma and burn induced HO [74,80]. Another model
assessed the expression of the inflammatory markers chemokine C-X-C
motif ligand 1 (CXCL1) and interleukin 6 (IL6) during the first 6 and 24 h
which showed an upregulation of these markers only following longer
duration blast waves while no changes were noted in short duration

6
Z. Kazezian and A.M.J. Bull Bone 143 (2021) 115765

blast waves [101]. An important investigation was done using rat blast Table 2
models to determine if the presence of bioburden (Acinetobacter bau­ In Vivo blast injury models developed to study HO.
mannii and methicillin-resistant Staphylococcus aureus (MRSA)) affects Blast injury Species Objectives Results Ref
the severity of the ectopic bone formation and whether there is any model
connection between bacterial contamination on the amputation site and Blast at a Rat 1. To determine the 1. The radiographic [39]
the HO formation [104]. These in vivo models of HO following blast magnitude onset of cartilage images showed
injury amputations are summarised in Table 2. Some of these models of 120 ± 7 and bone bone growth at
used blast and a drop weight tower to create the fracture as a separate kPa, development by 3–4 week time
femoral using points post-blast
insult following the blast overpressure phase [39,104–106]. Despite the fracture radiographs injury
fact that the majority of these published in vivo studies do not incorpo­ and 2. To identify 2. The genomic and
rate all three characteristics simultaneously, there is evidence that some quadriceps biomarkers of proteomic
are attempting to establish such models [107]. Although there are some crush by a osteogenic and analysis identified
drop chondrogenic a group of
models to study HO following blast injuries, it is clear that there is an
weight properties dysregulated
urgent need for new models that fully replicate the blast associated genes and key
amputation scenario through a single insult rather than using an extra osteogenic
tool to achieve the fracture of the bone. Once this need is addressed, proteins such as
models can be used to further our knowledge about other possible key RUNX-2, OCN,
NOG, SP-1 which
stimuli for blast associated HO development. were associated
with the early
5. Discussion development of
bone
Blast at a Rat 1. To investigate 1. None of the [105]
Although traumatic HO can arise from different types of mechani­
magnitude whether blast animals in the
cally induced trauma, it is only in recent decades that its high level of of 120 ± 7 overpressure group (BOP-CTL)
incidence post blast injury has become evident, most likely due to the kPa, increases the which were
very high levels of survival attained due to excellent medical care. femoral occurrence of HO exposed to blast
fracture after overpressure
Therefore, this review focuses mainly on the scientific research per­
and transfemoral alone developed
taining to traumatic HO and is focused on blast. quadriceps amputation HO
For HO to occur, the presence of the stem or progenitor cells is crush by a performed 2. 6/9 animals of the
essential. Some of the origins of these cells have been traced back to drop within the zone group (AMP-CTL)
mesenchymal [109], endothelium [67] and muscular origin [110], all of weight of injury by which undergone
exposing rats to crush and
which can differentiate into osteogenic cells. Although HO is clinically
a. blast amputation
characterized by bone formation which can be skeletally connected overpressure developed HO
[35], studies suggest that this kind of bone formation might have a (BOP-CTL) alone, that was detected
different pathway compared to the classical long bone formation during b. crush injury radiographically
embryogenesis which is mainly through endochondral ossification and femoral 3. All animals of the
fracture followed group (BOP-AMP)
[111,112]. Although there is still some contradiction regarding the exact by amputation with crush,
pathway of bone formation in HO [113–115], recent work has shown through the zone amputation and
through histological analysis that ectopic bone formed through HO can of injury (AMP- blast developed
be quite similar to fracture healing [113]; however, the mechanisms that CTL) or c. a HO
combination of a
control the fate of the progenitor cells contributing to HO are different
and b (BOP-
and are affected by different factors including the location of the stim­ AMP)
ulated progenitor cells [116,117]. Explosive Rat 1. To create a 1. 9/12 animals [102]
Understanding the molecular biomarkers and signalling pathways under survivable blast survived the
can help to identify new diagnostic tools in the clinic as well as therapies water injury model procedure
using water blast 2. 4 surviving rats
for blast induced HO. Several studies have explored some of these bio­ method in with hind-limb
markers [71–74]. The challenges in these studies are that early identi­ hindlimb/ amputation
fication of HO is necessary to explore the HO triggering pathways, yet forelimb of rats exhibited type A
this is not possible in the clinical situation specifically in blast injury (periosteal
growth) HO
associated HO. Therefore, there is a need to have appropriate in vivo
3. 3/4 also exhibited
models that allow this early phase to be investigated. Despite Inflam­ type B
mation being highlighted as a key driver in HO formation, mechanical (noncontiguous
loading has also been postulated as a main factor modulating HO [118] growth) HO
and research has found that mechanical loading can influence HO 4. 1/5 forelimb
amputees
through the mTORC1 (Fig. 2) signalling pathway, where in vivo studies exhibited types A
have shown that low elongation mechanical loading mitigated HO, and B HO
whilst high elongation mechanical loading augmented HO [118]. Explosive Rat 1. To create a 1. At 8 weeks, water [103]
Current in vivo HO models are limited in that bone fracture is not under sand survivable blast and sand blasted
or water injury model animals had a
always achieved as a result of the blast itself, but by using an extra tool
using water/sand median severity of
such as a drop weight [104–106]; this does not replicate the exact sit­ blast method in 1.0
uation/model of combat-related HO. There is no in vivo model that hindlimb of rats 2. At 16 weeks,
combines the blast and the fracture simultaneously in one insult, 2. To assess the water blasted
necessitating the development of more advanced models to better severity of HO animals had a
3. To assess the median severity of
replicate the disease conditions. This would enable the examination of type/location of 2.0 compared to
the early mechanisms producing HO to better understand the impact of HO 1.5 in the animals
the blast on the HO development. (continued on next page)
In conclusion, although blast associated HO is more prevalent than

7
Z. Kazezian and A.M.J. Bull Bone 143 (2021) 115765

Table 2 (continued ) Table 2 (continued )

Blast injury Species Objectives Results Ref Blast injury Species Objectives Results Ref
model model

that had been longer duration

sand blasted blast waves
3. At 24 weeks, 3. Regardless of the
animals that had pressure, no
been water significant
blasted had changes were
median severity of noted in short
2.5 compared to duration blast
2.0 in animals that wave
had been sand 4. No histological
blasted. No damage has been
significant noted to the liver,
difference was lung or muscles in
observed in HO all the settings
severity at any used in the
time point experiments
4. 6, 9, and 10 water- 5. All inflammatory
blasted animals at cytokines went
8, 16, and 24 back to the
weeks respec­ normal levels 24 h
tively were noted after blast
to have type 3 HO. Explosive Rat 1. To assess the 1. No significant [108]
Also, 7, 10, and 11 under efficacy of differences were
sand blasted ani­ water indomethacin found in the HO
mals, developed and irradiation to severity scores
type 3 HO at each hinder HO among the three
respective time stimulated by study groups
5. No significant high-energy blast (control (no
difference was injury treatment),
observed in HO Indomethacin,
type at any time and 8Gy
point radiation)
Blast at a Rat 1. To determine if 1. At 12 weeks, an [104] 2. In terms of
magnitude the presence of increased volume qualitative HO
of 120 ± 7 bioburden of HO was type scoring,
kPa, (Acinetobacter observed in rats indomethacin
femoral baumannii and infected with group was not
fracture MRSA) affects MRSA when significantly
and the severity of compared with A different than the
quadriceps the ectopic bone baumannii or control group
crush by a formation vehicle while the
drop 2. To determine the 2. Soft tissue and radiation group
weight persistent effect marrow of the had a higher HO
of bacterial residual limb of type score than
contamination rats inoculated those of
on the with A baumannii indomethacin and
amputation site were positive for the control
in relation to HO strains of bacteria Blast at a Rat 1. To investigate 1. Corticosterone [106]
formation colony-forming magnitude whether level was highest
units in bone of 120 kPa, corticosterone after 5 h post-
marrow and mus­ femoral and testosterone surgery in the
cle tissue, but fracture is associated with blast and injury
negative for A and ectopic bone group, after 24 h
baumannii quadriceps formation in in the injury only
infection crush by a blast associated and 5 days in the
3. Tissues from drop HO blast only groups.
MRSA-infected weight 2. Testosterone
rats were positive reached its lowest
for MRSA only in level post-
bone marrow and operation in a
muscle tissue similar fashion to
Shock tube 6 Rat 1. To investigate 1. At 6 h an elevation [101] the corticosterone
bar and 16 the acute in the level of increase demon­
bar (10% inflammatory neutrophils and strating the lowest
and 100% response to the monocytes were in the blast and
volume) blast wave in noted in rats injury group at 5
respect to exposed for longer h, the injury only
different duration blast group and 24 h,
magnitudes and waves and the blast only
duration 2. Increased group at 5 days
inflammatory 3. The amount of HO
cytokines such as formed detected
CXCL1 and IL6 at by CT scans at day
both 6 and 24 h in 40 post-operation
rats exposed for was highest in the
(continued on next page)

8
Z. Kazezian and A.M.J. Bull
Table 2 (continued )
Blast injury Species Objectives Results Ref
model
blast and injury
group, which was
followed by the
injury group.
other types of trauma induced HO, there is lack of understanding
whether the main driving force that pushes the fate of the progenitor
cells towards the osteogenic linage is the blast itself or the combination
of the blast and the trauma of the amputation. Although efforts have
been made to elucidate the rulers of these signalling pathways, there is
still a lack of information to be able to better diagnose and treat the
disease when focusing on the main drivers of HO specially after blast.
Next, we expect to see more preclinical blast injury models that can add
to our current information regarding the driver, cell origin, main trig­
gering pathway and specific targets of blast associated HO.
Funding
This work was supported by the Royal British Legion.
CRediT authorship contribution statement
Conceptualization and Writing (Original Draft): Zepur Kazezian
Writing, Reviewing & Editing: Anthony MJ Bull, Zepur Kazezian
Supervision: Anthony MJ Bull
Declaration of competing interest
The authors do not have any conflict of interest.
Acknowledgments
The authors would like to acknowledge the financial support of the
Royal British Legion.
References
[1] J.E. Puzas, M.D. Miller, R.N. Rosier, Pathologic bone formation, Clin. Orthop.
Relat. Res. 245 (1989) 269–281.
[2] B R. Demonstration line durch ach Hagiges Umhergehen total destruirten
kniegelenkes von einem patienten mit stichverletzing des ruckans. Verh Dtsch
Gesellschaft Chirurg 1883;12:93.
[3] C.A. Dejerine, Para-ostéo-arthropathies des paraplégiques par lésion médullaire
(étude clinique et radiographique), Ann Méd 5 (1918) 497–555.
[4] Dejerine CA, Dejerine Y Para-ostéo-arthropathies des paraplégiques par lésion
médullaire. Étude anatomique et histologique. Rev. Neurol. 1919;34:399–407.
[5] B.K. Potter, J.A. Forsberg, T.A. Davis, K.N. Evans, J.S. Hawksworth, D. Tadaki, et
al., Heterotopic ossification following combat-related trauma, J. Bone Joint Surg.
Am. 92 (Suppl. 2) (2010) 74–89.
[6] A. Martelli, A.R. Santos Jr., Cellular and morphological aspects of fibrodysplasia
ossificans progressiva. Lessons of formation, repair, and bone bioengineering,
Organogenesis. 10 (3) (2014) 303–311.
[7] R.J. Pignolo, G. Ramaswamy, J.T. Fong, E.M. Shore, F.S. Kaplan, Progressive
osseous heteroplasia: diagnosis, treatment, and prognosis, Appl. Clin. Genet. 8
(2015) 37–48.
[8] N.S. Adegbite, M. Xu, F.S. Kaplan, E.M. Shore, R.J. Pignolo, Diagnostic and
mutational spectrum of progressive osseous heteroplasia (POH) and other forms
of GNAS-based heterotopic ossification, Am. J. Med. Genet. A 146A (14) (2008)
1788–1796.
[9] Y. Zhu, F. Zhang, W. Chen, Q. Zhang, S. Liu, Y. Zhang, Incidence and risk factors
for heterotopic ossification after total hip arthroplasty: a meta-analysis, Arch.
Orthop. Trauma Surg. 135 (9) (2015) 1307–1314.
[10] S.N. Moore, G.D. Hawley, E.N. Smith, N.A. Mignemi, R.C. Ihejirika, M. Yuasa, et
al., Validation of a radiography-based quantification designed to longitudinally
monitor soft tissue calcification in skeletal muscle, PLoS One 11 (7) (2016),
e0159624.
[11] D.M. Tippets, A.V. Zaryanov, W.V. Burke, P.D. Patel, J.C. Suarez, E.E. Ely, et al.,
Incidence of heterotopic ossification in direct anterior total hip arthroplasty: a
retrospective radiographic review, J. Arthroplast. 29 (9) (2014) 1835–1838.
[12] W.T. Kent, T.J. Shelton, J. Eastman, Heterotopic ossification around the knee
after tibial nailing and ipsilateral antegrade and retrograde femoral nailing in the
treatment of floating knee injuries, Int. Orthop. 42 (6) (2018) 1379–1385.
9
Bone 143 (2021) 115765
[13] R.D. Brady, S.R. Shultz, S.J. McDonald, T.J. O’Brien, Neurological heterotopic
ossification: current understanding and future directions, Bone. 109 (2018)
35–42.
[14] B.K. Potter, T.C. Burns, A.P. Lacap, R.R. Granville, D.A. Gajewski, Heterotopic
ossification following traumatic and combat-related amputations. Prevalence,
risk factors, and preliminary results of excision, J. Bone Joint Surg. Am. 89 (3)
(2007) 476–486.
[15] D.E. Garland, C.E. Blum, R.L. Waters, Periarticular heterotopic ossification in
head-injured adults. Incidence and location, J. Bone Joint Surg. Am. 62 (7)
(1980) 1143–1146.
[16] R.H. Wittenberg, U. Peschke, U. Botel, Heterotopic ossification after spinal cord
injury. Epidemiology and risk factors, J. Bone Joint Surg. (Br.) 74 (2) (1992)
215–218.
[17] K.A. Alfieri, J.A. Forsberg, B.K. Potter, Blast injuries and heterotopic ossification,
Bone Joint Res. 1 (8) (2012) 192–197.
[18] K.V. Brown, S. Dharm-Datta, B.K. Potter, J. Etherington, A. Mistlin, J.R. Hsu, et
al., Comparison of development of heterotopic ossification in injured US and UK
armed services personnel with combat-related amputations: preliminary findings
and hypotheses regarding causality, J. Trauma 69 (Suppl. 1) (2010) S116–S122.
[19] Potter BK, Burns TC, Lacap AP, Granville RR, Gajewski D. Heterotopic ossification
in the residual limbs of traumatic and combat-related amputees. J Am Acad
Orthop Surg. 2006;14(10 Spec No.):S191–7.
[20] J.A. Forsberg, J.M. Pepek, S. Wagner, K. Wilson, J. Flint, R.C. Andersen, et al.,
Heterotopic ossification in high-energy wartime extremity injuries: prevalence
and risk factors, J. Bone Joint Surg. Am. 91 (5) (2009) 1084–1091.
[21] A. Ramasamy, W.G. Eardley, D.S. Edwards, J.C. Clasper, M.P. Stewart, Surgical
advances during the first world war: the birth of modern orthopaedics, J. R. Army
Med. Corps 162 (1) (2016) 12–17.
[22] G.J. Pavey, E.M. Polfer, K.E. Nappo, S.M. Tintle, J.A. Forsberg, B.K. Potter, What
risk factors predict recurrence of heterotopic ossification after excision in combat-
related amputations? Clin. Orthop. Relat. Res. 473 (9) (2015) 2814–2824.
[23] E.P. Hofmeister, M. Mazurek, J. Ingari, Injuries sustained to the upper extremity
due to modern warfare and the evolution of care, J. Hand. Surg. [Am.] 32 (8)
(2007) 1141–1147.
[24] D.C. Covey, Blast and fragment injuries of the musculoskeletal system, J. Bone
Joint Surg. Am. 84 (7) (2002) 1221–1234.
[25] Covey DC. Combat orthopaedics: a view from the trenches. J Am Acad Orthop
Surg. 2006;14(10 Spec No.):S10–7.
[26] C.K. Murray, J.R. Hsu, J.S. Solomkin, J.J. Keeling, R.C. Andersen, J.R. Ficke, et
al., Prevention and management of infections associated with combat-related
extremity injuries, J. Trauma 64 (3 Suppl) (2008) S239–S251.
[27] P.P. Alastair Beaven, Treatment principles of blast injuries, Surgery. 33 (2015) 9.
[28] Beaven A PP. Treatment principles of blast injuries. Surgery. 2015(33(9):424–9.
[29] A.J. Sheean, S.M. Tintle, P.C. Rhee, Soft tissue and wound management of blast
injuries, Curr Rev Musculoskelet Med. 8 (3) (2015) 265–271.
[30] C.A. de Candole, Blast injury, Can. Med. Assoc. J. 96 (4) (1967) 207–214.
[31] R.G. DePalma, D.G. Burris, H.R. Champion, M.J. Hodgson, Blast injuries, N. Engl.
J. Med. 352 (13) (2005) 1335–1342.
[32] J.A. Singleton, I.E. Gibb, A.M. Bull, P.F. Mahoney, J.C. Clasper, Primary blast
lung injury prevalence and fatal injuries from explosions: insights from
postmortem computed tomographic analysis of 121 improvised explosive device
fatalities, J. Trauma Acute Care Surg. 75 (2 Suppl 2) (2013) S269–S274.
[33] Lewis PC, Camou E, Wofford K. The impact of cigarette smoking on the formation
of heterotopic ossification among service members with a traumatic amputation.
Mil. Med. 2017;182(5):e1742-e8.
[34] G.J. Pavey, A.T. Qureshi, A.M. Tomasino, C.L. Honnold, D.K. Bishop, S. Agarwal,
et al., Targeted stimulation of retinoic acid receptor-gamma mitigates the
formation of heterotopic ossification in an established blast-related traumatic
injury model, Bone. 90 (2016) 159–167.
[35] D. Evriviades, S. Jeffery, T. Cubison, G. Lawton, M. Gill, D. Mortiboy, Shaping the
military wound: issues surrounding the reconstruction of injured servicemen at
the Royal Centre for Defence medicine, Philos. Trans. R. Soc. Lond. Ser. B Biol.
Sci. 366 (1562) (2011) 219–230.
[36] D. Shehab, A.H. Elgazzar, B.D. Collier, Heterotopic ossification, J. Nucl. Med. 43
(3) (2002) 346–353.
[37] K. Ranganathan, S. Loder, S. Agarwal, V.W. Wong, J. Forsberg, T.A. Davis, et al.,
Heterotopic ossification: basic-science principles and clinical correlates, J. Bone
Joint Surg. Am. 97 (13) (2015) 1101–1111.
[38] D.P. Clark, C.T. Badea, Micro-CT of rodents: state-of-the-art and future
perspectives, Phys Med. 30 (6) (2014) 619–634.
[39] A.T. Qureshi, E.K. Crump, G.J. Pavey, D.N. Hope, J.A. Forsberg, T.A. Davis, Early
characterization of blast-related heterotopic ossification in a rat model, Clin.
Orthop. Relat. Res. 473 (9) (2015) 2831–2839.
[40] T. Rosteius, E.M. Suero, D. Grasmucke, M. Aach, A. Gisevius, M. Ohlmeier, et al.,
The sensitivity of ultrasound screening examination in detecting heterotopic
ossification following spinal cord injury, Spinal Cord 55 (1) (2017) 71–73.
[41] Checa A. Ultrasonography, an operator-dependent modality versus dual-energy
computed tomography (DECT) in the detection of chondrocalcinosis: with regard
to Tanikawa et al.’s study. J Orthop Surg Res. 2018;13(1):255.
[42] A. Zagarella, E. Impellizzeri, R. Maiolino, R. Attolini, M.C. Castoldi, Pelvic
heterotopic ossification: when CT comes to the aid of MR imaging, Insights
Imaging. 4 (5) (2013) 595–603.
[43] E.M.W. Eekhoff, E. Botman, J. Coen Netelenbos, P. de Graaf, N. Bravenboer,
D. Micha, et al., [18F]NaF PET/CT scan as an early marker of heterotopic
ossification in fibrodysplasia ossificans progressiva, Bone. 109 (2018) 143–146.
Z. Kazezian and A.M.J. Bull
[44] J.E. Perosky, J.R. Peterson, O.N. Eboda, M.D. Morris, S.C. Wang, B. Levi, et al.,
Early detection of heterotopic ossification using near-infrared optical imaging
reveals dynamic turnover and progression of mineralization following Achilles
tenotomy and burn injury, J. Orthop. Res. 32 (11) (2014) 1416–1423.
[45] N.J. Crane, E. Polfer, E.A. Elster, B.K. Potter, J.A. Forsberg, Raman spectroscopic
analysis of combat-related heterotopic ossification development, Bone. 57 (2)
(2013) 335–342.
[46] A. Papour, J.H. Kwak, Z. Taylor, B. Wu, O. Stafsudd, W. Grundfest, Wide-field
Raman imaging for bone detection in tissue, Biomed Opt Express. 6 (10) (2015)
3892–3897.
[47] J.R. Peterson, P.I. Okagbare, S. De La Rosa, K.E. Cilwa, J.E. Perosky, O.N. Eboda,
et al., Early detection of burn induced heterotopic ossification using
transcutaneous Raman spectroscopy, Bone. 54 (1) (2013) 28–34.
[48] N.M. Eisenstein, S.C. Cox, R.L. Williams, S.A. Stapley, L.M. Grover, Bedside,
Benchtop, and bioengineering: physicochemical imaging techniques in
biomineralization, Adv Healthc Mater. 5 (5) (2016) 507–528.
[49] O.P. Gautschi, D. Cadosch, S.P. Frey, A.P. Skirving, L. Filgueira, R. Zellweger,
Serum-mediated osteogenic effect in traumatic brain-injured patients, ANZ J.
Surg. 79 (6) (2009) 449–455.
[50] B.Y. Klein, E. Shohami, Y. Reikhinshtein, H. Ben-Bassat, M. Liebergall, Serum-
mediated osteogenic effects of head injury on cultured rat marrow stromal cells,
Calcif. Tissue Int. 65 (3) (1999) 217–222.
[51] D. Cadosch, O.P. Gautschi, M. Thyer, S. Song, A.P. Skirving, L. Filgueira, et al.,
Humoral factors enhance fracture-healing and callus formation in patients with
traumatic brain injury, J. Bone Joint Surg. Am. 91 (2) (2009) 282–288.
[52] M. Boes, M. Kain, S. Kakar, F. Nicholls, D. Cullinane, L. Gerstenfeld, et al.,
Osteogenic effects of traumatic brain injury on experimental fracture-healing,
J. Bone Joint Surg. Am. 88 (4) (2006) 738–743.
[53] M.H. Kurer, M.A. Khoker, P. Dandona, Human osteoblast stimulation by sera from
paraplegic patients with heterotopic ossification, Paraplegia. 30 (3) (1992)
165–168.
[54] D. Cadosch, A.M. Toffoli, O.P. Gautschi, S.P. Frey, R. Zellweger, A.P. Skirving, et
al., Serum after traumatic brain injury increases proliferation and supports
expression of osteoblast markers in muscle cells, J. Bone Joint Surg. Am. 92 (3)
(2010) 645–653.
[55] K. Fujimura, K. Bessho, K. Kusumoto, Y. Konishi, Y. Ogawa, T. Iizuka,
Experimental osteoinduction by recombinant human bone morphogeneticprotein
2 in tissue with low blood flow: a study in rats, Br. J. Oral Maxillofac. Surg. 39 (4)
(2001) 294–300.
[56] D.S. Musgrave, P. Bosch, J.Y. Lee, D. Pelinkovic, S.C. Ghivizzani, J. Whalen, et al.,
Ex vivo gene therapy to produce bone using different cell types, Clin. Orthop.
Relat. Res. 378 (2000) 290–305.
[57] Y. Okubo, K. Bessho, K. Fujimura, Y. Konishi, K. Kusumoto, Y. Ogawa, et al.,
Osteoinduction by recombinant human bone morphogenetic protein-2 at
intramuscular, intermuscular, subcutaneous and intrafatty sites, Int. J. Oral
Maxillofac. Surg. 29 (1) (2000) 62–66.
[58] K. Yoshida, K. Bessho, K. Fujimura, K. Kusumoto, Y. Ogawa, Y. Tani, et al.,
Osteoinduction capability of recombinant human bone morphogenetic protein-2
in intramuscular and subcutaneous sites: an experimental study,
J. Craniomaxillofac. Surg. 26 (2) (1998) 112–115.
[59] M. Mastrogiacomo, A.R. Derubeis, R. Cancedda, Bone and cartilage formation by
skeletal muscle derived cells, J. Cell. Physiol. 204 (2) (2005) 594–603.
[60] D.S. Musgrave, R. Pruchnic, P. Bosch, B.H. Ziran, J. Whalen, J. Huard, Human
skeletal muscle cells in ex vivo gene therapy to deliver bone morphogenetic
protein-2, J. Bone Joint Surg. (Br.) 84 (1) (2002) 120–127.
[61] J.S. Sun, S.Y. Wu, F.H. Lin, The role of muscle-derived stem cells in bone tissue
engineering, Biomaterials. 26 (18) (2005) 3953–3960.
[62] Peterson JR, De La Rosa S, Eboda O, Cilwa KE, Agarwal S, Buchman SR, et al.
Treatment of heterotopic ossification through remote ATP hydrolysis. Sci Transl
Med. 2014;6(255):255ra132.
[63] F. Genet, I. Kulina, C. Vaquette, F. Torossian, S. Millard, A.R. Pettit, et al.,
Neurological heterotopic ossification following spinal cord injury is triggered by
macrophage-mediated inflammation in muscle, J. Pathol. 236 (2) (2015)
229–240.
[64] M.R. Convente, S.A. Chakkalakal, E. Yang, R.J. Caron, D. Zhang, T. Kambayashi,
et al., Depletion of mast cells and macrophages impairs heterotopic ossification in
an Acvr1(R206H) mouse model of fibrodysplasia ossificans progressiva, J. Bone
Miner. Res. 33 (2) (2018) 269–282.
[65] G. Chen, C. Deng, Y.P. Li, TGF-beta and BMP signaling in osteoblast
differentiation and bone formation, Int. J. Biol. Sci. 8 (2) (2012) 272–288.
[66] E. Leblanc, F. Trensz, S. Haroun, G. Drouin, E. Bergeron, C.M. Penton, et al., BMP-
9-induced muscle heterotopic ossification requires changes to the skeletal muscle
microenvironment, J. Bone Miner. Res. 26 (6) (2011) 1166–1177.
[67] V.Y. Lounev, R. Ramachandran, M.N. Wosczyna, M. Yamamoto, A.D. Maidment,
E.M. Shore, et al., Identification of progenitor cells that contribute to heterotopic
skeletogenesis, J. Bone Joint Surg. Am. 91 (3) (2009) 652–663.
[68] F.H. Gannon, F.S. Kaplan, E. Olmsted, G.C. Finkel, M.A. Zasloff, E. Shore, Bone
morphogenetic protein 2/4 in early fibromatous lesions of fibrodysplasia
ossificans progressiva, Hum. Pathol. 28 (3) (1997) 339–343.
[69] G. Grenier, E. Leblanc, N. Faucheux, D. Lauzier, P. Kloen, R.C. Hamdy, BMP-9
expression in human traumatic heterotopic ossification: a case report, Skelet.
Muscle 3 (1) (2013) 29.
[70] K. Tsuji, A. Bandyopadhyay, B.D. Harfe, K. Cox, S. Kakar, L. Gerstenfeld, et al.,
BMP2 activity, although dispensable for bone formation, is required for the
initiation of fracture healing, Nat. Genet. 38 (12) (2006) 1424–1429.
10
Bone 143 (2021) 115765
[71] K.N. Evans, B.K. Potter, T.S. Brown, T.A. Davis, E.A. Elster, J.A. Forsberg,
Osteogenic gene expression correlates with development of heterotopic
ossification in war wounds, Clin. Orthop. Relat. Res. 472 (2) (2014) 396–404.
[72] K.N. Evans, J.A. Forsberg, B.K. Potter, J.S. Hawksworth, T.S. Brown, R. Andersen,
et al., Inflammatory cytokine and chemokine expression is associated with
heterotopic ossification in high-energy penetrating war injuries, J. Orthop.
Trauma 26 (11) (2012) e204–e213.
[73] J.F. de Vasconcellos, S. Zicari, S.D. Fernicola, D.W. Griffin, Y. Ji, E.H. Shin, et al.,
In vivo model of human post-traumatic heterotopic ossification demonstrates
early fibroproliferative signature, J. Transl. Med. 17 (1) (2019) 248.
[74] Crowgey EL, Wyffels JT, Osborn PM, Wood TT, Edsberg LE. A systems biology
approach for studying heterotopic ossification: proteomic analysis of clinical
serum and tissue samples. genomics proteomics bioinformatics. 2018;16(3):
212–20.
[75] L.E. Edsberg, E.L. Crowgey, P.M. Osborn, J.T. Wyffels, A survey of proteomic
biomarkers for heterotopic ossification in blood serum, J. Orthop. Surg. Res. 12
(1) (2017) 69.
[76] J.M. Wilkinson, I. Stockley, A.J. Hamer, N.A. Barrington, R. Eastell, Biochemical
markers of bone turnover and development of heterotopic ossification after total
hip arthroplasty, J. Orthop. Res. 21 (3) (2003) 529–534.
[77] A.L. Sherman, J. Williams, L. Patrick, K. Banovac, The value of serum creatine
kinase in early diagnosis of heterotopic ossification, J Spinal Cord Med. 26 (3)
(2003) 227–230.
[78] L. Dong, G. Dong, J. Cao, J. Zhang, Association of alpha2-HS glycoprotein with
neurogenic heterotopic ossification in patients with spinal cord injury, Med. Sci.
Monit. 23 (2017) 5382–5388.
[79] S. Agarwal, S. Loder, C. Brownley, D. Cholok, L. Mangiavini, J. Li, et al.,
Inhibition of Hif1alpha prevents both trauma-induced and genetic heterotopic
ossification, Proc. Natl. Acad. Sci. U. S. A. 113 (3) (2016) E338–E347.
[80] L. Lin, Q. Shen, H. Leng, X. Duan, X. Fu, C. Yu, Synergistic inhibition of
endochondral bone formation by silencing Hif1alpha and Runx2 in trauma-
induced heterotopic ossification, Mol. Ther. 19 (8) (2011) 1426–1432.
[81] Y.K. Wang, W.F. Sun, X.G. Liu, J. Deng, B.E. Yan, W.Y. Jiang, et al., Comparative
study of serum levels of BMP-2 and heterotopic ossification in traumatic brain
injury and fractures patients, Zhongguo Gu Shang. 24 (5) (2011) 399–403.
[82] H. Kang, A.B. Dang, S.K. Joshi, B. Halloran, R. Nissenson, X. Zhang, et al., Novel
mouse model of spinal cord injury-induced heterotopic ossification, J. Rehabil.
Res. Dev. 51 (7) (2014) 1109–1118.
[83] Y. Nakamura, K. Tensho, H. Nakaya, M. Nawata, T. Okabe, S. Wakitani, Low dose
fibroblast growth factor-2 (FGF-2) enhances bone morphogenetic protein-2 (BMP-
2)-induced ectopic bone formation in mice, Bone. 36 (3) (2005) 399–407.
[84] S. Suutre, A. Toom, A. Arend, G. Selstam, Bone tissue content of TGF-beta2
changes with time in human heterotopic ossification after total hip arthroplasty,
Growth Factors 27 (2) (2009) 114–120.
[85] S. Gallea, F. Lallemand, A. Atfi, G. Rawadi, V. Ramez, S. Spinella-Jaegle, et al.,
Activation of mitogen-activated protein kinase cascades is involved in regulation
of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent
C2C12 cells, Bone. 28 (5) (2001) 491–498.
[86] C.F. Lai, S.L. Cheng, Signal transductions induced by bone morphogenetic
protein-2 and transforming growth factor-beta in normal human osteoblastic
cells, J. Biol. Chem. 277 (18) (2002) 15514–15522.
[87] B. Tu, S. Liu, G. Liu, Z. Li, Y. Sun, C. Fan, Inhibition of connexin 43 prevents
trauma-induced heterotopic ossification, Sci. Rep. 6 (2016) 37184.
[88] E.C. Martin, A.T. Qureshi, C.B. Llamas, E.C. Boos, A.G. King, P.C. Krause, et al.,
Trauma induced heterotopic ossification patient serum alters mitogen activated
protein kinase signaling in adipose stem cells, J. Cell. Physiol. 233 (9) (2018)
7035–7044.
[89] J. Wu, B. Ren, F. Shi, P. Hua, H. Lin, BMP and mTOR signaling in heterotopic
ossification: does their crosstalk provide therapeutic opportunities? J. Cell.
Biochem. 120 (8) (2019) 12108–12122.
[90] W.P. Tseng, S.N. Yang, C.H. Lai, C.H. Tang, Hypoxia induces BMP-2 expression
via ILK, Akt, mTOR, and HIF-1 pathways in osteoblasts, J. Cell. Physiol. 223 (3)
(2010) 810–818.
[91] Tu B, Liu S, Yu B, Zhu J, Ruan H, Tang T, et al. miR-203 inhibits the traumatic
heterotopic ossification by targeting Runx2. Cell Death Dis. 2016;7(10):e2436.
[92] X. Su, L. Liao, Y. Shuai, H. Jing, S. Liu, H. Zhou, et al., MiR-26a functions
oppositely in osteogenic differentiation of BMSCs and ADSCs depending on
distinct activation and roles of Wnt and BMP signaling pathway, Cell Death Dis. 6
(2015), e1851.
[93] M. Fransen, C. Anderson, J. Douglas, S. MacMahon, B. Neal, R. Norton, et al.,
Safety and efficacy of routine postoperative ibuprofen for pain and disability
related to ectopic bone formation after hip replacement surgery (HIPAID):
randomised controlled trial, BMJ. 333 (7567) (2006) 519.
[94] S. Winkler, F. Wagner, M. Weber, J. Matussek, B. Craiovan, G. Heers, et al.,
Current therapeutic strategies of heterotopic ossification–a survey amongst
orthopaedic and trauma departments in Germany, BMC Musculoskelet. Disord. 16
(2015) 313.
[95] R.J. Macfarlane, B.H. Ng, Z. Gamie, M.A. El Masry, S. Velonis, C. Schizas, et al.,
Pharmacological treatment of heterotopic ossification following hip and
acetabular surgery, Expert. Opin. Pharmacother. 9 (5) (2008) 767–786.
[96] S. Winkler, B. Craiovan, F. Wagner, M. Weber, J. Grifka, T. Renkawitz,
Pathogenesis and prevention strategies of heterotopic ossification in total hip
arthroplasty: a narrative literature review and results of a survey in Germany,
Arch. Orthop. Trauma Surg. 135 (4) (2015) 481–489.
[97] J.A. Aubut, S. Mehta, N. Cullen, R.W. Teasell, Group E, Research T. Scire,
A comparison of heterotopic ossification treatment within the traumatic brain
Z. Kazezian and A.M.J. Bull
and spinal cord injured population: an evidence based systematic review,
NeuroRehabilitation. 28 (2) (2011) 151–160.
[98] W.R. Barfield, R.E. Holmes, L.A. Hartsock, Heterotopic ossification in trauma,
Orthop Clin North Am. 48 (1) (2017) 35–46.
[99] G. Pavlou, M. Kyrkos, E. Tsialogiannis, N. Korres, E. Tsiridis, Pharmacological
treatment of heterotopic ossification following hip surgery: an update, Expert.
Opin. Pharmacother. 13 (5) (2012) 619–622.
[100] S.C. Land, A.R. Tee, Hypoxia-inducible factor 1alpha is regulated by the
mammalian target of rapamycin (mTOR) via an mTOR signaling motif, J. Biol.
Chem. 282 (28) (2007) 20534–20543.
[101] T. Eftaxiopoulou, A. Barnett-Vanes, H. Arora, W. Macdonald, T.T. Nguyen,
M. Itadani, et al., Prolonged but not short-duration blast waves elicit acute
inflammation in a rodent model of primary blast limb trauma, Injury. 47 (3)
(2016) 625–632.
[102] O. Tannous, C. Griffith, R.V. O’Toole, V.D. Pellegrini Jr., Heterotopic ossification
after extremity blast amputation in a Sprague-Dawley rat animal model,
J. Orthop. Trauma 25 (8) (2011) 506–510.
[103] D.E. Jaffe, D. Yoo, J. Blevins, G. Gasbarro, T. Hughes, E. Paryavi, et al., Does blast
medium affect heterotopic ossification in a blast-amputation model? Clin. Orthop.
Relat. Res. 473 (8) (2015) 2680–2687.
[104] G.J. Pavey, A.T. Qureshi, D.N. Hope, R.L. Pavlicek, B.K. Potter, J.A. Forsberg, et
al., Bioburden increases heterotopic ossification formation in an established rat
model, Clin. Orthop. Relat. Res. 473 (9) (2015) 2840–2847.
[105] E.M. Polfer, D.N. Hope, E.A. Elster, A.T. Qureshi, T.A. Davis, D. Golden, et al., The
development of a rat model to investigate the formation of blast-related post-
traumatic heterotopic ossification, Bone Joint J. 97-B (4) (2015) 572–576.
[106] M.Y. Qin, R.E. Atwood, W.A. Ketchum, S.A. Kaba, M.J. Bradley, Characterization
of serum levels of testosterone and corticosterone in a blast and amputation rat
model of heterotopic ossification, Mol. Cell. Endocrinol. 509 (2020) 110799.
[107] D.L. Williams, R.T. Epperson, N.B. Taylor, M.B. Nielsen, B.S. Kawaguchi, D.
L. Rothberg, et al., System setup to deliver air impact forces to a sheep limb:
preparation for model development of blast-related heterotopic ossification, JMIR
Res Protoc. 8 (2) (2019), e12107.
11
Bone 143 (2021) 115765
[108] A.D. Robertson, A.M. Chiaramonti, T.P. Nguyen, D.E. Jaffe, R.E. Holmes, E.
L. Hanna, et al., Failure of indomethacin and radiation to prevent blast-induced
heterotopic ossification in a Sprague-Dawley rat model, Clin. Orthop. Relat. Res.
477 (3) (2019) 644–654.
[109] L. Kan, C.Y. Peng, T.L. McGuire, J.A. Kessler, Glast-expressing progenitor cells
contribute to heterotopic ossification, Bone. 53 (1) (2013) 194–203.
[110] B.G. Matthews, E. Torreggiani, E. Roeder, I. Matic, D. Grcevic, I. Kalajzic,
Osteogenic potential of alpha smooth muscle actin expressing muscle resident
progenitor cells, Bone. 84 (2016) 69–77.
[111] O. Tannous, A.C. Stall, C. Griffith, C.T. Donaldson, R.J. Castellani Jr., V.
D. Pellegrini Jr., Heterotopic bone formation about the hip undergoes
endochondral ossification: a rabbit model, Clin. Orthop. Relat. Res. 471 (5)
(2013) 1584–1592.
[112] T. Stoeger, G.E. Proetzel, H. Welzel, A. Papadimitriou, C. Dony, R. Balling, et al.,
In situ gene expression analysis during BMP2-induced ectopic bone formation in
mice shows simultaneous endochondral and intramembranous ossification,
Growth Factors 20 (4) (2002) 197–210.
[113] K.L. Foley, N. Hebela, M.A. Keenan, R.J. Pignolo, Histopathology of periarticular
non-hereditary heterotopic ossification, Bone. 109 (2018) 65–70.
[114] J.R. Peterson, S. Agarwal, R.C. Brownley, S.J. Loder, K. Ranganathan, P.
S. Cederna, et al., Direct mouse trauma/burn model of heterotopic ossification,
J. Vis. Exp. 102 (2015), e52880.
[115] B.H. Dubansky, B.D. Dubansky, Natural development of dermal ectopic bone in
the american alligator (Alligator mississippiensis) resembles heterotopic
ossification disorders in humans, Anat Rec (Hoboken). 301 (1) (2018) 56–76.
[116] L. Kan, Y. Liu, T.L. McGuire, D.M. Berger, R.B. Awatramani, S.M. Dymecki, et al.,
Dysregulation of local stem/progenitor cells as a common cellular mechanism for
heterotopic ossification, Stem Cells 27 (1) (2009) 150–156.
[117] M. Sorkin, A.K. Huber, C. Hwang, Wft Carson, R. Menon, J. Li, et al., Regulation
of heterotopic ossification by monocytes in a mouse model of aberrant wound
healing, Nat. Commun. 11 (1) (2020) 722.
[118] G. Chen, H. Jiang, X. Tian, J. Tang, X. Bai, Z. Zhang, et al., Mechanical loading
modulates heterotopic ossification in calcific tendinopathy through the mTORC1
signaling pathway, Mol. Med. Rep. 16 (5) (2017) 5901–5907.