Animal models of typical heterotopic ossification.
ABSTRACT Heterotopic ossification (HO) is the formation of marrow-containing bone outside of the normal skeleton. Acquired HO following traumatic events is a common and costly clinical complication. In contrast, hereditary HO is rarer, progressive, and life-threatening. Substantial effort has been directed towards understanding the mechanisms underlying HO and finding efficient treatments. However, one crucial limiting factor has been the lack of relevant animal models. This article reviews the major currently available animal models, summarizes some of the insights gained from these studies, and discusses the potential future challenges and directions in HO research.
- SourceAvailable from: Katherine E Cilwa[Show abstract] [Hide abstract]
ABSTRACT: OBJECTIVE:: To demonstrate the pro-osteogenic effect of burn injury on heterotopic bone formation using a novel burn ossicle in vivo model. BACKGROUND:: Heterotopic ossification (HO), or the abnormal formation of bone in soft tissue, is a troubling sequela of burn and trauma injuries. The exact mechanism by which burn injury influences bone formation is unknown. The aim of this study was to develop a mouse model to study the effect of burn injury on heterotopic bone formation. We hypothesized that burn injury would enhance early vascularization and subsequent bone formation of subcutaneously implanted mesenchymal stem cells. METHODS:: Mouse adipose-derived stem cells were harvested from C57/BL6 mice, transfected with a BMP-2 adenovirus, seeded on collagen scaffolds (ossicles), and implanted subcutaneously in the flank region of 8 adult mice. Burn and sham groups were created with exposure of 30% surface area on the dorsum to 60°C water or 30°C water for 18 seconds, respectively (n = 4/group). Heterotopic bone volume was analyzed in vivo by micro-computed tomography for 3 months. Histological analysis of vasculogenesis was performed with platelet endothelial cell adhesion molecule staining. Osteogenic histological analysis was performed by Safranin O, Picrosirius red, and aniline blue staining. Qualitative analysis of heterotopic bone composition was completed with ex vivo Raman spectroscopy. RESULTS:: Subcutaneously implanted ossicles formed heterotopic bone. Ossicles from mice with burn injuries developed significantly more bone than sham control mice, analyzed by micro-computed tomography at 1, 2, and 3 months (P < 0.05), and had enhanced early and late endochondral ossification as demonstrated by Safranin O, Picrosirius red, and aniline blue staining. In addition, burn injury enhanced vascularization of the ossicles (P < 0.05). All ossicles demonstrated chemical composition characteristic of bone as demonstrated by Raman spectroscopy. CONCLUSIONS:: Burn injury increases the predilection to osteogenic differentiation of ectopically implanted ossicles. Early differences in vascularity correlated with later bone development. Understanding the role of burn injury on heterotopic bone formation is an important first step toward the development of treatment strategies aimed to prevent unwanted and detrimental heterotopic bone formation.Annals of surgery 05/2013; · 7.90 Impact Factor
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ABSTRACT: Proliferation and fusion of myoblasts is a well-orchestrated process occurring during muscle development and regeneration. Although myoblasts are known to originate from muscle satellite cells, the molecular mechanisms that coordinate their commitment toward differentiation are poorly understood. Here, we present a novel role for the transcription factor Forkhead box protein C2 (Foxc2) in regulating proliferation and preventing premature differentiation of activated muscle satellite cells. We demonstrate that Foxc2 expression is upregulated early in activated mouse muscle satellite cells and then diminishes during myogenesis. In undifferentiated C2C12 myoblasts, downregulation of endogenous Foxc2 expression leads to a decrease in proliferation, whereas forced expression of FOXC2 sustains proliferation and prevents differentiation into myotubes. We also show that FOXC2 induces Wnt signaling by direct interaction with the Wnt4 (wingless-type MMTV integration site family member-4) promoter region. The resulting elevated expression of bone morphogenetic protein-4 (Bmp4) and RhoA-GTP proteins inhibits the proper myoblast alignment and fusion required for myotube formation. Interestingly, continuous forced expression of FOXC2 alters the commitment of C2C12 myoblasts toward osteogenic differentiation, which is consistent with FOXC2 expression observed in patients with myositis ossificans, an abnormal bone growth within muscle tissue. In summary, our results suggest that (a) Foxc2 regulates the proliferation of multipotent muscle satellite cells; (b) downregulation of Foxc2 is critical for myogenesis to progress; and (c) sustained Foxc2 expression in myoblast cells suppresses myogenesis and alters their lineage commitment toward osteogenesis by inducing the Wnt4 and Bmp4 signaling pathways.Cell Death and Differentiation advance online publication, 3 May 2013; doi:10.1038/cdd.2013.34.Cell death and differentiation 05/2013; · 8.24 Impact Factor
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ABSTRACT: Celecoxib, a selective cox-2 inhibitor, has been shown to prevent the heterotopic ossification following total hip arthroplasty. However, the effects of celecoxib on heterotopic ossification at other locations remain unclear. This study aimed to investigate the effect of celecoxib on heterotopic ossification in the rat model with Achilles tenotomy. Forty male Sprague-Dawley rats, which were randomly divided into 2 groups (n = 20), underwent midpoint Achilles tenotomy on left legs through a posterior approach under aseptic condition. Experimental group was treated with the saline solution of celecoxib (10 mg/kg) per day, while control group was treated by normal saline (0.9%). At 3, 5 and 10 postoperative weeks, all animals were examined by X-ray to assess new bone formation in the Achilles tendon. At 10 weeks after surgery, all animals were killed and Achilles tendons were taken for hematoxylin-eosin (HE) and immunohistochemical staining. Heterotopic ossification developed in 3 rats (15%) in experimental group and 20 rats (100%) in control group by postoperative 10 weeks. The incidence of heterotopic ossification was significantly lower in experimental group than in control group (P < 0.05). Our findings suggest that celecoxib inhibits HO development in rat model with Achilles tenotomy.European Journal of Orthopaedic Surgery & Traumatology 02/2013; 23(2):145-8. · 0.18 Impact Factor
Hindawi Publishing Corporation
Journal of Biomedicine and Biotechnology
Volume 2011, Article ID 309287, 8 pages
AnimalModelsof TypicalHeterotopic Ossification
LixinKan andJohn A.Kessler
Department of Neurology, Northwestern University Feinberg Medical School, 303 East Chicago Avenue, Chicago, IL 60611, USA
Correspondence should be addressed to Lixin Kan, email@example.com
Received 26 August 2010; Accepted 28 September 2010
Academic Editor: Monica Fedele
Copyright © 2011 L. Kan and J. A. Kessler. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Heterotopic ossification (HO) is the formation of marrow-containing bone outside of the normal skeleton. Acquired HO
following traumatic events is a common and costly clinical complication. In contrast, hereditary HO is rarer, progressive, and
life-threatening. Substantial effort has been directed towards understanding the mechanisms underlying HO and finding efficient
treatments. However, one crucial limiting factor has been the lack of relevant animal models. This article reviews the major
currently available animal models, summarizes some of the insights gained from these studies, and discusses the potential future
challenges and directions in HO research.
Heterotopic ossification (HO) is the formation of marrow-
containing bone outside of the normal skeleton [1, 2].
Acquired HO following traumatic events, such as total joint
replacements (TJR) [3–5], spinal cord injury (SCI) ,
traumatic brain injury (TBI) , fracture, muscular trauma,
or war-wounded patients [8, 9], is a common and costly
clinical complication. Hereditary HO, such as fibrodysplasia
ossificans progressiva (FOP), is rare, progressive, and life
threatening . The first description of hereditary HO in
FOP was made in 1692 by Guy Patin. Acquired HO as a
complication of gunshot wounds was described by Dejerine
and Ceillier in 1918 . 16%–53% of SCI (11,000 annually)
and TBI (1.4 million) patients and 40%–50% of TJR (1
million) patients will develop HO at some point.
in range of motion. Once acquired HO develops, surgical
removal is the only effective treatment, normally followed
by local radiation or nonsteroidal anti-inflammatory agents
(NSAIDs) to prevent recurrence . However, surgical
removal is costly, the effectiveness of NSAIDs is variable,
and radiation has been associated with malignancies [13,
14]. Further, there is no effective treatment for debilitating
hereditary HO, FOP .
Substantial effort has been directed towards understand-
ing the mechanisms underlying HO and finding efficient
treatments. However, one crucial limiting factor has been
the lack of relevant animal models. This paper reviews
the long and arduous efforts to generate clinical relevant
available models. It also summarizes some of the insights
gained from these studies and discusses the potential future
challenges and directions in HO research.
For the purposes of this paper, HO is defined as a hetero-
geneous disorder characterized by pathologic endochondral
ossification with hematopoietic bone marrow in soft tissues,
suchassubcutaneoustissue,skeletal muscle, or fibroustissue
adjacent to joints. Similar pathologies lacking endochondral
ossification, such as Progressive Osseous Heteroplasia [15,
16] or that containing no hematopoietic bone marrow,
such as ectopic calcification/mineralization  (also called
dystrophic calcification), are not included.
2.AnimalModels of HereditaryHO
A typical example of hereditary HO is FOP, which is char-
acterized by stereotyped patterned progressive ossification
in soft tissues . In this disorder, mutations in a bone
morphogenetic protein (BMP) receptor gene, ACVR1 ,
result in dysregulation of BMP signaling that ultimately
leads to FOP [10, 20]. This mutant ACVR1 activates BMP
signaling in the absence of BMP ligand leading to BMP-
independent chondrogenesis that is enhanced by BMP
2Journal of Biomedicine and Biotechnology
ligands . This indicates that mechanism-based animal
models that faithfully replicate the disorder virtually require
genetic modifications leading to enhanced BMP signaling.
Theoretically, there are at least five ways to modify and
enhance BMP signaling: (1) by introducing a hyperactive
BMP receptor, (2) by knocking out BMP inhibitors, (3)
by introducing high level of BMPs, (4) by overexpressing
specific BMP target genes, and (5) by modifying BMP
signaling indirectly through other factors that can interact
with components of BMP signaling pathway.
Animal models that introduce hyperactive BMP receptor,
especially the recently found mutations in ACVR1, would
seem to be the most relevant model of FOP. However,
introducing a constitutively active ACVR1 mutation into
zebrafish embryos failed to induce obvious HO even though
strong ventralization due to enhanced BMP signaling was
observed . A genetically modified mouse model that
carries the same mutation has not yet been reported but
will likely be a valuable addition to the field in the future.
Interestingly, a number of animals including domestic cats
[22–25], shepherd dog , pigs , and the Southeast
Asian mouse deer of the genus Tragulus  develop
FOP-like conditions spontaneously. Even though the exact
genetic bases are still unknown, it is reasonable to think
that sporadic, spontaneous mutations in the BMP signaling
pathway, especially mutations in ACVR1, were likely respon-
sible for the observed FOP-like conditions. Further genetic
studies of these affected animals hopefully will clarify this
issue. However, due to the rarity of the events and ethical
considerations, the practicality of these large animal models
as a drug testing platform is in question.
Kobayashi et al.  reported that Col2-caBmpr1a
transgenic mice that express constitutive active Bmpr1a
(caBmpr1a) under the control of rat type II collagen
promoter created enhanced BMP signaling. E17.5 transgenic
embryos showed severe skeletal abnormalities; the femur,
tibia, and patella were fused together, eliminating joint tis-
sues . This study demonstrated that overactive BMP sig-
drocyte maturation toward hypertrophic differentiation, but
an HO phenotype was not reported in this model. Fukuda et
al. reported using a Cre-loxP system to conditionally express
a constitutively active ALK2 receptor (caALK2) to activate
BMP signaling, but this produced embryonic lethality .
Their data indicate that low levels of caAlk2 expression are
sufficient to transduce a sufficient amount of BMP signaling
to compromise normal development of embryos. We specu-
late that conditionally activating the caAlk2 expression with
late tissue-specific Cre in future studies might generate a
mouse model that is useful for study of HO.
2.2. Animal Models That Knock out BMP Inhibitors. Genetic
BMP signaling and potentially producing an animal model
with an FOP-like phenotype. In fact, Noggin-/- mice have
some congenital skeletal defects, including congenital HO,
but Noggin-/- mice die soon after birth . Mice with
targeted disruption of Chordin, another BMP inhibitor, also
die at birth, and they develop defects in inner and outer
ear development and show abnormalities in pharyngeal
and cardiovascular organization . Mutation of another
mouse competitive BMP inhibitor gene, Gremlin, resulted
also in a severe abnormal skeletal pattern . Interestingly,
conditional deletion of Gremlin by crossing the floxed mice
with osteocalcin promoter-driven cre (Oc-Cre) caused only
a transient increase in bone formation and bone mass, but
not HO . Null mutation of less specific inhibitors of
BMP signaling, such as Dan  or Cerberus-like , did
not generate gross defects; these two mutant lines are born
alive and fertile without a postnatal HO phenotype. Thus,
although null mutation of genes encoding BMP inhibitors
provided insights into how enhanced BMP signaling affects
embryonic development, especially skeletal development,
none of the mutant mouse lines generated by this strategy
are useful for postnatal HO studies. This likely reflects the
pleiotropic roles of BMP signaling in various tissues.
2.3. Animal Models That Overexpress BMP Ligand. The
rationale for enhancing BMP signaling by overexpressing
BMP ligand is straightforward, but this approach has met
a number of unexpected complications. Overexpression of
BMP4 under the control of many different promoters does
not lead to postnatal HO. For example, HO does not develop
after overexpression of BMP4 under control of either the
keratin promoter (K14)  or the bovine cytokeratin IV
promoter . Transgenic mice overexpressing BMP4 under
control of surfactant protein-C gene promoter die from
abnormally formed lungs . Transgenic mice expressing
BMP4 in cartilage under the control of the Col11a2 pro-
moter/enhancer sequences die at birth due to respiratory
failure  while mice overexpressing human BMP4 under
control of mouse Msx1 minimal promoter develop no
visible abnormalities . Overexpression of BMP2 under
the human αSM-actin promoter in an ApoE-deficient back-
ground accelerates atherosclerotic intimal calcification in
transgenic lines but does not produce typical HO . Mice
that overexpress BMP4 under the Nephrin promoter have
interesting defects in glomerular capillary formation but not
the HO phenotype .
The only exception has been mice that overexpress
(Nse-BMP4). These mice develop a phenotype that closely
recapitulates the FOP phenotype and that also displays the
histological hallmarks of typical acquired HO . These
findings suggest that overexpression of BMP itself may be
necessary but is not sufficient to generate the HO phenotype
and that the correct expression patterns or contexts are
crucial. We have extensively characterized the phenotype in
this transgenic mouse line and have used these mice, in
collaboration with other labs , to study different aspects
of HO, including definition of the events that trigger HO,
the type of cells that respond to the trigger by differentiating
the spread of HO .
Journal of Biomedicine and Biotechnology3
2.4. Animal Models That Overexpress a Specific BMP Target
Gene. If overexpression of BMP ligand can produce HO, it
is reasonable to think that expressing specific BMP target
genes might also be capable of copying the phenotype of
BMP overexpression. In fact, overexpression of MSX2, a
BMP target gene, can induce an HO-like phenotype. MSX2
an ubiquitous promoter, such as CMV, a tissue specific pro-
moter, such as TIMPl (tissue inhibitor of metalloproteinase
1), or the endogenous MSX2 promoter [35, 62]. Overex-
pression of another BMP target gene, Runx2, under the
type II collagen promoter also caused an HO-like phenotype
and ectopic expression of hypertrophic chondrocyte markers
. These two models show that both MSX2 and Runx2
can partially mediate the osteogenic effects of BMPs in vivo.
However, since the phenotypes in these two lines do not
closely mimic that of FOP, the relevance of these models
to the human disease is still unclear. Moreover, multiple
transgenic lines that overexpress other BMP target genes,
especially the Id family genes, that is, Id1-Id4 [63–65], have
failed to produce an HO phenotype. This could be partially
explained by the inadequate tissue specific promoters used in
generating these transgenic lines. However, the failure more
mediating the HO phenotype, even though Id1 and Id3 are
positive factors in promotion of bone formation in vivo .
2.5. Animal Models That Overexpress Other Factors That
Indirectly Modify the BMP Signaling Pathway. Theoretically,
it is also possible to enhance the BMP signaling indirectly
through factors that can interact with components of the
BMP signaling pathway. For example, overexpression of
Fos in bone cells under control of an FBJ long terminal
repeat element (H2-FosLTR) resulted in the development of
calcified tumors similar to HO, and Fos-ES cell chimeras
developed chondrosarcomas with high efficiency at all skele-
tal sites containing cartilage . However, transgenic mice
that overexpress other related AP1 members (e.g., JUN and
FOSB) do not exhibit abnormalities, despite high expression
in bone tissue. Not surprisingly, further studies provided
evidence of specific interactions between the BMP-signaling
pathway and c-Fos, but not the other related AP1 members
in FOP-like lesions .
Overall, even though there are multiple ways to enhance
BMP signaling in vivo, only a few genetic modified animal
lines showed typical HO, or a phenotype resembling FOP.
Further, only one line, Nse-BMP4 transgenic mice, closely
recapitulated the major aspects of the FOP phenotype.
3.AnimalModels for AcquiredHO
Acquired HO usually follows traumatic events, such as
fracture, total hip arthroplasty, muscular trauma, spinal cord
injury, or central nervous system injury. It is a relatively
frequent clinical complication with a wide clinical spectrum
but normally it has a relatively benign course . The etiol-
ogy of common acquired HO is still unclear, and multiple
contributing factors have been proposed including BMPs,
inflammation, prostaglandin E2, hypercalcemia, hypoxia,
abnormal nerve activities, immobilization, and disequilib-
rium of hormones [66, 67]. Lack of deep understanding
of underlying molecular mechanisms has directly hindered
the validation of existing animal models, and this also has
limited the development of new mechanism-based animal
models. Currently, there are several available animal models
that can produce typical HO: (1) heterotopic implantation
models, (2) hip arthroplasty model, (3) the immobilization
manipulation model (also called the Michelsson model), (4)
Achilles tenotomy model, (5) trauma-induced model, and
(6) models generated by injection of irritants and other
materials to muscle.
3.1. Heterotopic Implantation Model. Currently the most
commonly used animal model for HO involves the surgical
implantation of BMP containing matrix at heterotopic sites.
Implantation of demineralized bone matrix was first used
by Urist in 1965 ; then Wozney et al. were able to
repeat the experiment using partially purified BMP proteins
. Currently, the most widely used approach is BMP
matrigel implantation ; an advantage of this method
is that a chilled mix can be injected into heterotopic sites
as a liquid which gels on site at body temperature and
thereafter releases BMP4 continuously at the site. Many
modifications/variations of this method have been used
in different species under different conditions, including
introduction of a DNA construct that produces BMPs
, microbubble-enhanced transcutaneous sonoporation
of human BMP2 , nanogel-cross-linking hydrogel as
a scaffold , implantation of a slow-release system of
6Al-4V implants coated with native BMPs .
One interesting variation on this theme involves direct
injection into the heterotopic site of cells that have
osteogenic, and/or osteogenic factor producing potential,
such as bone marrow cells , or implantation of a diffu-
sion chamber containing such cells. Tested cell types have
included urinary tract epithelia , certain transformed
cells such as transformed human amnion cells (FL cells)
, Moloney sarcoma , and epithelial-like cells .
In a similar system, these cells are impregnated into ceramic
blocks to test their osteogenic activity  in the presence or
absence of an osteogenic inducer.
Another interesting approach takes advantage of the
osteoinductive ability of certain biomaterials, such as micro-
porous calcium phosphate ceramic particles , that do
not release BMP or other known osteogenic factors. The
mechanism of osteoinduction by such biomaterials is not
currently clear, although the geometry of the material is
thought to be important .
Generally, heterotopic implantation models are straight-
forward, repeatable, and mechanistically relevant to human
HO. However, certain limitations do exist: (1) they are
artificial systems that may create unphysiologically high
local concentrations of osteogenic factors in implanted sites
leading to effects not relevant to the human disorder, (2) the
implantation is a local event and thus has limited ability to
mimic the potential effects of the involvement of multiple
4Journal of Biomedicine and Biotechnology
systems, (3) different variations of this method have variable
reliabilities and relevance to human conditions, (4) the
incidence of implantation-induced bone formation varies
depending upon the material or animal species. Normally
rabbits are the most, and mice the least, susceptible , and
experimental conditions that produce ectopic bone do not
always coincide with clinical observations in humans.
3.2. Hip Arthroplasty Model. HO is commonly observed
after hip arthroplasty in humans for unknown reasons. To
develop a model relevant to the human condition ,
Schneider et al. subjected rabbits to surgery analogous to
human hip arthroplasty either with or without muscle and
of postoperative radiation in prophylaxis of HO was then
analyzed using this model. The rationale behind this model
is straightforward, and it can produce HO with certain
reliability; however, despite being a phenocopy of the human
condition, it is not a mechanism-based model. This method
has not been widely adapted by other investigators, probably
due to the relatively complicated surgical procedure.
3.3. The Michelsson Model (Also Called Immobilization
Manipulation Model). Michelsson et al.  found that
repeated forced mobilization of an immobilized knee joint
caused HO in the quadriceps muscle in rabbits, and similar
procedures can induce HO around other joints in the
rabbit as well. The precise inductive stimulus has not been
identified in this model, but an interaction between the
periosteum and the necrotic muscle seems necessary since
the introduction of a plastic membrane between bone and
muscle prevents bone formation . The first sign of
osteoblastic activity was seen in the periosteum, and the
new bone was often formed in continuity with the perios-
teum. Interestingly, early changes in prostaglandins preceded
bone formation , consistent with the hypothesis that
inflammation is the basis of the heterotopic bone formation
in that process. Several authors have used this model to
study the development and prevention of HO in animals
[81–85]. However, since HO in this model is not affected
by denervation, in contradistinction to clinical findings in
patients with neurologic injuries, the relevance of this model
to human HO is unclear.
3.4. Achilles Tenotomy Model. The Achilles tenotomy model
was first described in rats by Buck in 1953 , and in
1983, McClure applied the model to mice and found that
ectopic bone developed in 60% of animals by 5 weeks
and in 100% by 10 weeks after Achilles tenotomy .
The advantages of this model are its relative simplicity and
excellent predictability. However, the molecular mechanisms
of HO induced by Achilles tenotomy are poorly understood,
and the relevance of this model to clinical conditions is also
unclear since ectopic bone formation in Achilles tendon is a
associated with prior surgery or trauma to the tendon but is
also an important manifestation of rheumatoid arthritis and
ankylosing spondylitis .
3.5. Trauma-Induced Models. Traumatic muscle or CNS
injury often leads to HO in humans, but the underlying
causative factor(s) remains unknown. Efforts to establish
trauma induced models have had only limited success.
Zaccalini and Urist failed to induce HO in rabbit thigh
by blunt force . Walton et al. reported limited success
in inducing HO in sheep thigh by repeated blunt force (7
out of 42 sheep) . Further, intramembranous and not
endochondral ossification was the histological feature within
scar tissue. Based on these reports, these models do not
seem to be sufficiently reliable to be used routinely. Further,
the failure of this strategy has forced us to rethink why
trauma, which clearly plays a role in human HO, does not
using Nse-BMP4 mice have demonstrated that mild trauma
leads to HO with high frequency irrespective of which limb
is injured. In turn this suggests that trauma-induced HO
depends upon susceptibility determined by other factors—
in this case elevated levels of BMP4. The high frequency and
reproducibility of trauma-induced HO in this model may
provide a means of exploring the underlying mechanisms.
3.6. Irritant and Other Miscellaneous Material-Induced Mod-
els. Injection of various irritant materials into muscle some-
times leads to HO. For example, Heinen et al. reported the
induction of HO in rabbit by injection of 40% ethanol .
Selle and Urist also reported that acid-alcohol could induce
HO in a small percent of animals, while injections of calcium
chloride produced only amorphous calcified plaques, not
new bone or cartilage . In addition, Arai et al. 
and Caselli et al.  reported a controversial finding that
colchicine induced intramedullary bone formation. This
finding could not be repeated by K. H. Wlodarski and
P. Wlodarski , and later Dudkiewicz et al. found that
colchicine actually inhibits HO in a rabbit model . The
issues of repeatability and relevance of these models to
human HO limits their potential utility.
Overall, due to limited understanding of molecular
mechanisms, most animal models for acquired HO can only
mimic some aspects of the human conditions. Further, the
reliability and questionable clinical relevance hinder their
use as drug test platforms. Thus caution must be taken
in choosing one of these models to be appropriate for the
specific question being asked.
Multiple animal models have been generated for studies of
HO (see Table 1). For the simplicity of description in this
review, we divided these models into two major groups,
is arbitrary since injury and inflammation facilitates and
triggers HO in FOP as well as in animal models of hereditary
HO, and the high variability in susceptibility of different
individuals to acquired HO suggests a genetic basis for
individual predisposition. In fact, accumulating clinical and
experimental evidence suggests that similar cellular and
molecular mechanisms underlie the pathophysiology of all
Journal of Biomedicine and Biotechnology5
Table 1: Summary of commonly used animal models.
General strategy examplesHO related phenotypes?
Animal models of
Hyperactive BMP receptor
caALK2 in mice
Embryonic lethal, no HO
Knocking out BMP
Postnatal lethal, skeletal
Postnatal lethal, skeletal
K14-BMP4 No HO 
Msx1-BMP4 No observable defects
ProgressiveHO, FOP-like 
Overexpressing BMP target
Calcified tumors similar to
Modifying BMP signaling
Fos-ES cell chimeras
Animal models of acquired
BMP matrigel implantationHO 
DNA construct that
hydrogel as a scaffold
Bone marrow cells
Hip arthroplasty modelHip arthroplasty in rabbits
manipulation in rabbits
Michelsson modelHO 
Achilles tenotomy modelAchilles tenotomy in ratsHO
Blunt force in rabbit thigh
Repeated blunt force in
HO in small % of treated
Inject irritants and other
Injection of 40% ethanol HO
HO in small % of treated
Injection of acid-alcohol
typical HO which involves formation of fibroproliferative
lesions containing cells that follow the classic endochondral
ossification pathway. Thus, in hereditary HO, a specific
genetic mutation plays the centralrole, while in acquired HO
reason, some animal models such as Nse-BMP4 mice can be
used to study both hereditary and acquired HO.
Understanding the fundamental pathophysiology under-
lying HO is the key to development of mechanism-based
animal models. Just as determination of the genetic basis of
FOP opened up a whole new avenue for generating models
for hereditary HO, deeper understanding of the molecular
mechanisms underlying acquired HO will lead to more
fruitful approaches in generating new animal models for
the disorder. Multiple contributing factors are necessary for
acquired HO including a trigger (trauma, injury), osteogenic
progenitor cells, and a permissive microenvironment. How-
ever, thus far there is no single hypothesis that integrates
most clinical and experimental findings, and current data
strongly suggests the involvement of multiple organ systems
in this disorder. For this reason, future multidisciplinary
studies of neuroimmunological interactions and osteoneu-
roimmunology using currently available animal models,
such as Nse-BMP4 mice, will be necessary to provide the
6Journal of Biomedicine and Biotechnology
new insights which in turn could lay the foundation for new
mechanism-based animal models.
The authors thank Dr. Frederick Kaplan, who has been an
inspiration to us and to the entire field, for his advice,
encouragement, and support. This paper was supported in
part by Grants to Lixin Kan from the Center for Research in
FOP and Related disorders of the University of Pennsylvania
School of Medicine. John A Kessler is supported by the NIH
Grants nos. R01 020013-25 and R01 020778-25.
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