Understanding heterotopic ossification after spinal cord injury

Kulina, Irina (2015). Understanding heterotopic ossification after spinal cord injury PhD Thesis, School of Medicine, The University of Queensland. doi:10.14264/uql.2015.753

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Author Kulina, Irina
Thesis Title Understanding heterotopic ossification after spinal cord injury
School, Centre or Institute School of Medicine
Institution The University of Queensland
DOI 10.14264/uql.2015.753
Publication date 2015-06-26
Thesis type PhD Thesis
Supervisor Jean-Pierre Levesque
Ingrid Winkler
Total pages 193
Language eng
Subjects 1103 Clinical Sciences
1109 Neurosciences
1107 Immunology
Formatted abstract
Neurological heterotopic ossification (NHO) is a frequent complication of spinal cord and traumatic brain injuries (15-25% of patients) and manifests as abnormal ossification of soft tissues near joints. NHO is very debilitating and further delays rehabilitation as it causes pain, joint deformation and ankylosis and vascular and nerve compression. In the absence of an animal model the mechanisms leading to NHO are unknown and consequently there is no preventive treatment. The only effective approach to eliminate HO is complicated and expensive surgical resection. However these surgeries are delicate and can be challenging as they are performed once the NHO are mature, often large and incapacitating entrapping large blood vessels and nerves.

To elucidate NHO pathophysiology, we have developed the first animal model of NHO in genetically unmodified mice. Mice underwent a spinal cord transection (SCI); muscular inflammation was induced by intramuscular injection of cardiotoxin in limbs (IM-CTX). Formation of NHO was followed by μCT and immunohistology.

SCI alone or muscular inflammation alone did not induce NHO in mice. The combination of both SCI and muscular inflammation was necessary to induce NHO. This is consistent with clinical observations as NHO incidence is higher in patients with severe trauma or concomitant infection. Abundant F4/80+macrophages, which can provide pro-anabolic support in bone formation, were detected within the inflamed muscle and associated with areas of intramuscular bone formation (confirmed by von Kossa and collagen type 1 staining). In vivo depletion of phagocytic macrophages with clodronate-loaded liposomes prevented NHO formation whereas Zoledronate treatment exacerbated NHO. This supports our hypothesis that macrophage-mediated inflammation is a key activator of NHO following SCI. To further identify the source and type of macrophages, involved in the bone formation after SCI and test some potential treatment options different knock-out mouse models were investigated. My data suggest that local muscle residential macrophages potentially play an important role in NHO.

In addition we identified several populations of muscle progenitor cells that are prone to osteogenic differentiation in vitro. These data suggest that the mesenchymal progenitors that differentiate into osteoblasts in HO following spinal cord injury are already present in healthy muscles and therefore can be derived from local muscle progenitor cells rather than being recruited from the remote site, such as bone marrow.

Finally we investigated why SCI was necessary for NHO development. My hypothesis was that SCI causes release of systemic factors priming NHO. In support of this, I showed that NHO developed in non-paralysed inflamed front limbs of mice with SCI. Also, blood plasma from mice with SCI and IM-CTX induced osteogenic differentiation of cultured muscle mesenchymal progenitor cells (mMPC) sorted from naïve mice. This suggests that systemic factors facilitating NHO, such as substance P and G-CSF are released following SCI. These factors were then specifically targeted them in our mouse model to trial different types of treatment for prevention of bone formation.

In conclusion, our model suggests that NHO is a 2-insult process with 1) SCI inducing the release of factors that sensitize muscle progenitor cells to abnormal osteogenic differentiation and 2) macrophages accumulating in inflamed muscles then triggering abnormal osteogenic differentiation of mMPC. This study represents a significant advance in the understanding of NHO, revealing two targetable pathophysiologic mechanisms.
Keyword Spinal cord injury
Heterotopic ossification
Macrophage
Bone Formation
Inflammation

Document type: Thesis
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Created: Thu, 11 Jun 2015, 20:33:30 EST by Irina Kulina on behalf of Scholarly Communication and Digitisation Service