Sensory nerve induced inflammation contributes to heterotopic ossification

Abstract

Heterotopic ossification (HO), or bone formation in soft tissues, is often the result of traumatic injury. Much evidence has linked the release of BMPs (bone morphogenetic proteins) upon injury to this process. HO was once thought to be a rare occurrence, but recent statistics from the military suggest that as many as 60% of traumatic injuries, resulting from bomb blasts, have associated HO. In this study, we attempt to define the role of peripheral nerves in this process. Since BMP2 has been shown previously to induce release of the neuroinflammatory molecules, substance P (SP) and calcitonin gene related peptide (CGRP), from peripheral, sensory neurons, we examined this process in vivo. SP and CGRP are rapidly expressed upon delivery of BMP2 and remain elevated throughout bone formation. In animals lacking functional sensory neurons (TRPV1−/−), BMP2-mediated increases in SP and CGRP were suppressed as compared to the normal animals, and HO was dramatically inhibited in these deficient mice, suggesting that neuroinflammation plays a functional role. Mast cells, known to be recruited by SP and CGRP, were elevated after BMP2 induction. These mast cells were localized to the nerve structures and underwent degranulation. When degranulation was inhibited using cromolyn, HO was again reduced significantly. Immunohistochemical analysis revealed nerves expressing the stem cell markers nanog and Klf4, as well as the osteoblast marker osterix, after BMP2 induction, in mice treated with cromolyn. The data collectively suggest that BMP2 can act directly on sensory neurons to induce neurogenic inflammation, resulting in nerve remodeling and the migration/release of osteogenic and other stem cells from the nerve. Further, blocking this process significantly reduces HO, suggesting that the stem cell population contributes to bone formation.

Progenitors in Peripheral Nerves Launch Heterotopic Ossification

“…Since these cells are behind the blood-nerve barrier [26], it is difficult to envision how BMP2 enters this location. However, the timing appears to be similar to induction of the neuro-inflammatory pathways previously described [12, 14]. The lack of PS + cells in the soft tissues outside the nerve may be a direct result of rapid binding of BMP2 by inhibitory binding proteins in the region [27].…”

“…In addition to accessory cells migrating from the perineurial layer, cells within the endoneurium or axon compartment were found to express the osteoblast-specific factors SP7 and Dlx5 [12] [13]. These SP7 + Dlx5 + cells exit the nerve through the endoneurial vessels and are deposited at the site of new bone formation [7].…”

Botulinum Toxin-induced Muscle Paralysis Inhibits Heterotopic Bone Formation

“…Given the known mechanism of action of BTxA (inhibition of neurotransmitter release), we therefore speculated that the blockade of neuromuscular signaling by BTxA inhibits neuromuscular interactions integral to the osteogenic response elicited by musculoskeletal trauma. Furthermore, because fracture healing and HO share common initiating events (including inflammation and BMP signaling) [17,27,30,32], we also speculated that BTxA-induced muscle paralysis would prevent the formation of heterotopic bone. However, interpreting the precise mechanism by which muscle paralysis inhibits osteogenesis in the rat fracture model is confounded by the heterogeneity of the tissue response, soft tissue injury, and modified gait kinematics, all of which can alter the observed cellular responses.…”

Anaplerotic Accumulation of Tricarboxylic Acid Cycle Intermediates as Well as Changes in Other Key Metabolites During Heterotopic Ossification

“…However, recent studies have shown that the polyamines spermine and spermidine are an essential part of the granule component of mast cells, and play an essential role in the biogenesis and homeostasis of these organelles [Garcia‐Faroldi et al, 2010]. We have previously shown that mast cells are a critical component of HO and when these cells are eliminated HO is severely inhibited [Salisbury et al, 2011]. Therefore, spermine and spermidine may participate in the function of mast cells during HO.…”

“…This model involves both changes in BMP signaling, because of BMP2 production, as well as inflammation because of delivery of the adenovirus‐transduced cells. Characterization of this model, shows one of the first changes in the tissues is in peripheral nerves [Salisbury et al, 2011, 2012; Lazard et al, 2015], followed by the release of cells from the perineurial layer of the adjacent peripheral nerves of cells that express uncoupling protein 1 (UCP1) [Olmsted‐Davis et al, 2007]. UCP1 is thought to be the hallmark of brown adipocytes, and further characterization of these cells suggest they match almost identically brown adipocytes present in depots, with the exception that they lack expression of PMDR16 [Salisbury et al, 2012].…”