Lymphatic Contribution to the Cellular Niche in Heterotopic Ossification.

OBJECTIVE: The objective of this study was to determine the contribution of lymphatic tissue to heterotopic ossification (HO). BACKGROUND: HO is the pathologic development of ectopic bone within soft tissues often following severe trauma. Characterization of the tissue niche supporting HO is critical to identifying therapies directed against this condition. Lymphangiogenesis is upregulated during incidents of trauma, thereby coincident with the niche supportive of HO. We hypothesized that lymphatic tissues play a critical role in HO formation. METHODS: Mice underwent hindlimb Achilles' tendon transection and dorsal burn injury (burn/tenotomy) to induce HO. The popliteal and inguinal lymph nodes were excised ipsilateral to the tenotomy site. Flow cytometry and immunostaining were used to quantify and localize lymphoendothelium. MicroCT was used to quantify HO. RESULTS: Enrichment of mature lymphatic tissues was noted 2 weeks after injury at the tendon transection sites when compared with the contralateral, intact tendon based on LYVE1+ tubules (10.9% vs 0.8%, P < 0.05). Excision of the inguinal and popliteal nodes with draining popliteal lymphatic vessel significantly decreased the presence of mature lymphoendothelium 2 weeks after injury (10.9% vs 3.3%, P < 0.05). Bone-cartilage-stromal progenitor cells (CD105+/AlphaV+/Tie2-/CD45-/CD90-/BP1-) were also significantly decreased after lymph node excision (10.2% vs 0.5%, P < 0.05). A significant decrease was noted in the volume of de novo HO present within the soft tissues (0.12 mm vs 0.02 mm). CONCLUSION: These findings suggest that lymphatic vessels are intimately linked with the de novo formation bone within soft tissues following trauma, and their presence may facilitate bone formation.

Surgical Excision of Heterotopic Ossification Leads to Re-Emergence of Mesenchymal Stem Cell Populations Responsible for Recurrence.

Trauma-induced heterotopic ossification (HO) occurs after severe musculoskeletal injuries and burns, and presents a significant barrier to patient rehabilitation. Interestingly, the incidence of HO significantly increases with repeated operations and after resection of previous HO. Treatment of established heterotopic ossification is challenging because surgical excision is often incomplete, with evidence of persistent heterotopic bone. As a result, patients may continue to report the signs or symptoms of HO, including chronic pain, nonhealing wounds, and joint restriction. In this study, we designed a model of recurrent HO that occurs after surgical excision of mature HO in a mouse model of hind-limb Achilles' tendon transection with dorsal burn injury. We first demonstrated that key signaling mediators of HO, including bone morphogenetic protein signaling, are diminished in mature bone. However, upon surgical excision, we have noted upregulation of downstream mediators of osteogenic differentiation, including pSMAD 1/5. Additionally, surgical excision resulted in re-emergence of a mesenchymal cell population marked by expression of platelet-derived growth factor receptor-α (PDGFRα) and present in the initial developing HO lesion but absent in mature HO. In the recurrent lesion, these PDGFRα+ mesenchymal cells are also highly proliferative, similar to the initial developing HO lesion. These findings indicate that surgical excision of HO results in recurrence through similar mesenchymal cell populations and signaling mechanisms that are present in the initial developing HO lesion. These results are consistent with findings in patients that new foci of ectopic bone can develop in excision sites and are likely related to de novo formation rather than extension of unresected bone. Stem Cells Translational Medicine 2017;6:799-806.

Evaluation of Salivary Cytokines for Diagnosis of both Trauma-Induced and Genetic Heterotopic Ossification.

PURPOSE: Heterotopic ossification (HO) occurs in the setting of persistent systemic inflammation. The identification of reliable biomarkers can serve as an early diagnostic tool for HO, especially given the current lack of effective treatment strategies. Although serum biomarkers have great utility, they can be inappropriate or ineffective in traumatic acute injuries and in patients with fibrodysplasia ossificans progressiva (FOP). Therefore, the goal of this study is to profile the cytokines associated with HO using a different non-invasive source of biomarkers. METHODS: Serum and saliva were collected from a model of trauma-induced HO (tHO) with hind limb Achilles' tenotomy and dorsal burn injury at indicated time points (pre-injury, 48 h, 1 week, and 3 weeks post-injury) and a genetic non-trauma HO model (Nfatc1-Cre/caAcvr1fl/wt ). Samples were analyzed for 27 cytokines using the Bio-Plex assay. Histologic evaluation was performed in Nfatc1-Cre/caAcvr1fl/wt mice and at 48 h and 1 week post-injury in burn tenotomy mice. The mRNA expression levels of these cytokines at the tenotomy site were also quantified with quantitative real-time PCR. Pearson correlation coefficient was assessed between saliva and serum. RESULTS: Levels of TNF-α and IL-1ß peaked at 48 h and 1 week post-injury in the burn/tenotomy cohort, and these values were significantly higher when compared with both uninjured (p < 0.01, p < 0.03) and burn-only mice (p < 0.01, p < 0.01). Immunofluorescence staining confirmed enhanced expression of IL-1ß, TNF-α, and MCP-1 at the tenotomy site 48 h after injury. Monocyte chemoattractant protein-1 (MCP-1) and VEGF was detected in saliva showing elevated levels at 1 week post-injury in our tHO model when compared with both uninjured (p < 0.001, p < 0.01) and burn-only mice (p < 0.005, p < 0.01). The Pearson correlation between serum MCP-1 and salivary MCP-1 was statistically significant (r = 0.9686, p < 0.001) Similarly, the Pearson correlation between serum VEGF and salivary VEGF was statistically significant (r = 0.9709, p < 0.05). CONCLUSION: In this preliminary study, we characterized the diagnostic potential of specific salivary cytokines that may serve as biomarkers for an early-stage diagnosis of HO. This study identified two candidate biomarkers for further study and suggests a novel method for diagnosis in the context of current difficult diagnosis and risks of current diagnostic methods in certain patients.