BMP-9 expression in human traumatic heterotopic ossification: a case report
© Grenier et al.; licensee BioMed Central Ltd. 2013
Received: 6 October 2013
Accepted: 20 November 2013
Published: 16 December 2013
Heterotopic ossification (HO) is defined as the abnormal formation of mature bone in soft tissue, notably skeletal muscle. The morbidity of HO in polytraumatized patients impacts the functional outcome, impairs rehabilitation, and increases costs due to subsequent surgical interventions.
We present the case of a 34-year-old African male who developed severe HO around his right hip 11 days after a major trauma. Immunohistochemical analyses of resected tissue revealed that several BMPs were expressed in the HO, including highly osteogenic BMP-9.
To the best of our knowledge, this is the first report of local BMP expression, notably BMP-9, in traumatic HO, and suggests that BMP-9, possibly through mrSCs, can contribute to HO formation in soft tissues when a suitable microenvironment is present.
KeywordsBMP-9 Traumatic heterotopic ossification Muscle resident stromal cells
Heterotopic ossification (HO) involves ectopic bone formation in soft tissues such as muscles and is often associated with trauma . While the etiology of HO has been classified as neurogenic, traumatic, and hereditary, the exact pathophysiology of traumatic HO remains unknown. However, several critical factors such as progenitor cell populations, inductive factors, and a permissive environment may contribute to HO [2, 3].
BMPs play a critical role in the osteoblastic commitment of mesenchymal cells and the induction of osteoblastic activity [4, 5], and are potential candidates as inductive HO factors. BMPs are members of the transforming growth factor-beta (TGF-β) family . More than 20 BMPs have been described to date, but experimental evidence indicates that only BMP-2, -6, -7, and -9 can induce osteogenesis, BMP-9 being one of the most potent osteogenic BMPs [7, 8]. In addition, we recently reported that BMP-9 only induces HO in damaged muscle in a murine model . Despite the fact that BMP-9 transcripts are barely detectable in human skeletal muscle , no studies have examined the expression of BMPs, including BMP-9, in human HO.
The BMP pathway is regulated by a negative feedback mechanism involving extracellular inhibitors such as noggin, chordin, and gremlin, membrane pseudo-receptors such as BAMBI, and the intracellular inhibitors Smad-6 and Smad-7 [11, 12]. Based on studies by our group and others on the role of BMPs in normal and pathologic bone healing [13–17], we hypothesized that the balance between BMPs and their inhibitors may play a key role in the development of traumatic human HO.
In the present report, we describe the case of a patient with severe lower extremity trauma who developed HO. We determined the locations of BMPs, extracellular BMP antagonists, and BMP receptor BMPR1a by immunohistochemical staining. We propose a mechanism to explain the pathogenesis of trauma-associated HO and provide a novel perspective on the involvement of members of the BMP family.
A 34 year-old African male was brought to the emergency department after jumping from the seventh floor of an apartment building during a police raid. Physical and radiological examinations revealed a C1 fracture, a carpal dislocation, and a T-type fracture of the right acetabulum.
On the day of admission (Day 0), an open reduction and internal fixation (ORIF) of the acetabular fracture was performed using a standard Kocher-Langenbeck approach associated with a trochanteric flip .
A revision for suboptimal reduction was performed on Day 10, followed by a second stage ORIF the next day using an ilioinguinal approach . During the revision surgery, a mass of heterotopic bone was resected and sent for analysis. The patient did not receive NSAIDs or radiotherapy preoperatively. Clinical and radiological evaluations were performed for three and a half months postoperatively.
The tissue specimen harvested during the second surgery (Day 11) was processed for immunohistochemical and histological analyses.
Histology and immunohistochemistry
The sample was fixed in buffered formalin overnight, decalcified in 10% EDTA (pH 7.2) for three weeks, embedded in paraffin, and sectioned using a Leica RM 2255 microtome (Leica Microsystems, Richmond Hill, ON, Canada). Following deparaffinization and hydration, the tissue sections were stained with Goldner’s trichrome to visualize nuclei (blue-gray), cartilage (red-purple), osteoid (orange-red), and mineralized bone (green).
Primary and secondary antibodies used for the immunohistochemical staining procedure
Biotinylated horse anti-goat IgG
Biotinylated goat anti-rabbit IgG
Biotinylated goat anti-mouse IgG
Grading of the immunostained sections
Immunohistochemical staining of cellular and extracellular proteins has been used in mandibular , long bone distraction osteogenesis [21–24], and bone healing [13–15] studies. We employed this technique to semi-quantitatively evaluate our results based on the percentage of positively stained cells using the following grading scheme: +, 25% of the cells stained positively for the protein of interest; ++, 25 to 50% of the cells stained positively; +++, 50 to 75% of the cells stained positively; ++++, more than 75% of the cells stained positively; -, no cells stained positively.
Radiological and clinical follow-up
Macroscopic and microscopic tissue organization
Expression of BMPs, BMP receptors and their antagonists
Immunolocalization of BMP receptors, agonists, and antagonists
The difference in BMP expression in healing fractures and in HO might also be explained by the nature of the cells involved. A number of studies using animal models [9, 31] and human cells  have shown that muscle resident stromal cells (mrSCs) with high osteogenic potential contribute to HO. This finding has important clinical implications since mrSCs may be involved in fracture repair [33–35]. More importantly, non-unions are more likely to be associated with bones with minimal soft tissue coverage such as the tibia, and it has been proposed that non-unions could occur due to the lack of enveloping muscle tissue . The present case report underlines the importance of concentrating future studies on identifying the true role of multipotent mrSCs and the pathway leading to their osteogenic commitment.
We showed that BMPs are expressed in traumatic HO and that BMP-9 in particular may play a pivotal role in HO because of the more permissive environment of damaged muscle and the inability of BMP inhibitors in the microenvironment to alter the osteogenic program of multipotent progenitor cells. We propose that osteogenic multipotent progenitor cells, including mrSCs, and their signaling pathways should be investigated further with a view to developing prophylactic measures to prevent traumatic HO.
Informed consent was obtained from the patient for the publication of this case report and any accompanying images.
bone morphogenetic protein
muscle resident stromal cell
non-steroidal anti-inflammatory drugs
open reduction and internal fixation
transforming growth factor-beta.
We are grateful to Dr. Amy Svotelis for her critical reading of the manuscript. GG and RH received investigator awards from Fonds de Recherche Québec en Santé (FRQS). GG, NF, and RH hold grants from the Canadian Institutes of Health Research (CIHR).
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