C. Hwang1, C. A. Kubiak1, M. Sorkin1, C. A. Pagani1, T. Rehse1, M. A. Garada1, Z. N. Khatib1, P. Kotha1, J. Lisiecki1, D. M. Stepien1, N. D. Visser1, K. Vasquez1, A. W. James1, Y. S. Niknafs1, P. S. Cederna1, S. W. Kemp1, B. Levi1 1University Of Michigan,Surgery,Ann Arbor, MI, USA
Introduction: Heterotopic ossification (HO) is a painful, debilitating formation of ectopic bone, often found after severe polytrauma, burn and neural injury. Literature has implicated neurotrophic signals such as nerve growth factor (NGF) as crucial signals for normal bone development. Additionally, secreted neural peptides including substance P (SP) have been demonstrated as capable of regulating osteoblast behavior, modulating osteogenic cues, and producing ectopic bone when exogenously introduced. Thus, we hypothesized that innervation is crucial to pathologic stem cell differentiation and resultant HO.
Methods: C57BL/6J male mice were stratified to burn/tenotomy (BT) or BT+neurectomy. The Achilles tendon was bisected and accompanied by 30% TBSA dorsal burn. Sciatic/sural nerves were transected at midthigh (proximal/distal to bifurcation, respectively). Hindlimbs were analyzed via µCT at 9 weeks (n=3-5/group). Sections (n=1) from the ankle were immunolabeled (IF) at 1 and 3 weeks. Myeloperoxidase (MPO) activity was measured via in vivo imaging system at the ankle at 4 days (n=4-5/group). From BT mice, mRNA was harvested from injured and uninjured tendon (n=3/group) and hybridized to Affymetrix microarray (1 week) or processed for whole transcriptome via RNAseq (3 weeks). Microarray data of human ligament cells from GEO dataset GSE5464 were analyzed using linear modeling with empirical Bayes method for differential expression.
Results: Sciatic neurectomy upon BT significantly reduced total HO (Fig A, 4.7 v. 1.57mm3, p=0.036). IF imaging of NGF and SP showed robust protein expression at 1 week with limited colocalization of F4/80 or Ly6G (Fig. B,C). Neurectomies did not cause changes in MPO levels (Fig. D). BT (Fig. E, right) induced upregulation of characteristic genes of inflammation (Il6, Ptgs2, Ptger1, Il1a, Tacr1) vs. ininjured tendon at 1 week (left). Similar upregulation was observed at 3 weeks (Fig. F). Notable neurotrophins, Ngf, Gdnf, and Brdf, were also upregulated. GEO data exhibited parallel trends of Ngf and Brdf in human spinal ligament cells subjected to cyclic strain (Fig. G); a loading shown to be associated with ossification of connective tissues.
Conclusion: Interruption of innervation to an injury site inhibits post-traumatic ossification independent of myeloid cell infiltration (MPO) during the acute response. In mice, BT induces upregulation of neurotrophin genes in both acute and sub-acute timepoints, concordant with upregulation seen in strained human cells. BT exhibited robust labeling of NGF, spatially distinct from macrophages and PMNs, along with SP, a well characterized regulator of osteogenesis. This data suggests neural signals modulate aberrant wound healing as demonstrated by HO formation.
