Caitlin Priest

mTOR inhibition and BMP signaling act synergistically to reduce muscle fibrosis and improve myofiber regeneration

Muscle trauma is highly morbid due to intramuscular scarring, or fibrosis, and muscle atrophy. Studies have shown that bone morphogenetic proteins (BMPs) reduce muscle atrophy. However, increased BMP signaling at muscle injury sites causes heterotopic ossification, as seen in patients with fibrodysplasia ossificans progressiva (FOP), or patients with surgically placed BMP implants for bone healing. We use a genetic mouse model of hyperactive BMP signaling to show the development of intramuscular fibrosis surrounding areas of ectopic bone following muscle injury. Rapamycin, which we have previously shown to eliminate ectopic ossification in this model, also eliminates fibrosis without reducing osteogenic differentiation, suggesting clinical value for patients with FOP and with BMP implants. Finally, we use reporter mice to show that BMP signaling is positively associated with myofiber cross-sectional area. These findings underscore an approach in which 2 therapeutics (rapamycin and BMP ligand) can offset each other, leading to an improved outcome.

High-frequency spectral ultrasound imaging (SUSI) visualizes early post-traumatic heterotopic ossification (HO) in a mouse model

PURPOSE Early treatment of heterotopic ossification (HO) is currently limited by delayed diagnosis due to limited visualization at early time points. In this study, we validate the use of spectral ultrasound imaging (SUSI) in an animal model to detect HO as early as one week after burn tenotomy. METHODS Concurrent SUSI, micro CT, and histology at 1, 2, 4, and 9weeks post-injury were used to follow the progression of HO after an Achilles tenotomy and 30% total body surface area burn (n=3-5 limbs per time point). To compare the use of SUSI in different types of injury models, mice (n=5 per group) underwent either burn/tenotomy or skin incision injury and were imaged using a 55MHz probe on VisualSonics VEVO 770 system at one week post injury to evaluate the ability of SUSI to distinguish between edema and HO. Average acoustic concentration (AAC) and average scatterer diameter (ASD) were calculated for each ultrasound image frame. Micro CT was used to calculate the total volume of HO. Histology was used to confirm bone formation. RESULTS Using SUSI, HO was visualized as early as 1week after injury. HO was visualized earliest by 4weeks after injury by micro CT. The average acoustic concentration of HO was 33% more than that of the control limb (n=5). Spectroscopic foci of HO present at 1week that persisted throughout all time points correlated with the HO present at 9weeks on micro CT imaging. CONCLUSION SUSI visualizes HO as early as one week after injury in an animal model. SUSI represents a new imaging modality with promise for early diagnosis of HO.

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-1β 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-1β, 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.

Abstract 46. Modulating TAK1 Signaling to Enhance Scaffold and Cell-Free Calvarial Healing

T ueday, M arch 8, 2017 METHODS: One hundred fifty-eight patients with 277 expanded skin cases during 2010 to 2014 were reviewed and photograph-evaluated for the expanded skin texture and regenerative condition. Overall texture of the expanded skin flaps (Good, Fair, Poor) were evaluated and documented by senior attending surgeons. The occurrence of five indications of skin regeneration limitation, including skin thickness, skin color, stretch mark, vessel varicose and skin lesion, during skin expansion were recorded. The correlation of indications to overall skin regeneration condition was statistically analyzed.

Abstract 2: Macrophage-specific TGF-B is a Targetable Cytokine to Prevent Heterotopic Ossification

PURPOSE: Lymphedema is a common, life-long complication of cancer treatment that currently has no cure. Patients with lymphedema have decreased quality of life and suffer recurrent infections, while current treatments are merely palliative and designed to prevent disease progression. Accumulating evidence indicates that T cells play a key role in the pathology of lymphedema by inhibiting lymphangiogenesis and promoting tissue fibrosis. Because the pathophysiology of lymphedema involves primarily the skin and subcutaneous tissues, it may be possible to target T cells locally using topical medications such as tacrolimus without inducing systemic immunosuppression. The purpose of this study was therefore to study the efficacy of topical tacrolimus for prevention and treatment of lymphedema using preclinical mouse models.

Abstract 10: Use of a Novel MicroRNA-519c Construct for Targeted Downregulation of Hypoxia Inducible Factor 1 (Hif-1α)

PURPOSE: Heterotopic Ossification (HO) is the abnormal formation of ectopic bone after trauma or thermal injury. Hypoxia-inducible factor-1 alpha (Hif-1α) is a critical mediator of this process. We have previously demonstrated that loss of Hif-1α in a transgenic mouse model decreases HO. Building on these findings, the goal of the current study is to use targeted inhibition of Hif-1α to decrease HO formation. MicroRNAs (miRNA) are emerging in the field of cancer research as an important method of specific and targeted intervention given their unique ability to modulate protein translation without the severe side effects common to more ubiquitous modalities of treatment. In this study, we introduce the novel formulation of miRNA within a polymer sphere vehicle as a therapeutic option for HO that improves delivery and selectivity compared to other available alternatives. We validate the efficacy of this novel polymermicroRNA polyplex to downregulate Hif-1α and vascular endothelial growth factor (VEGF) to serve as a potential pharmacologic antagonist against HO in a targeted fashion.

Abstract 16: Inhibition of mTOR Signaling Reduces Mesenchymal Cell Migration To Sites Of Injury And Eliminates Heterotopic Ossification In A Mouse Model

INTRODUCTION: Several experimental studies suggest that growth hormone-based therapies have the potential to accelerate and augment axonal regeneration while simultaneously act directly on muscle and Schwann cells (SCs) to minimize denervation atrophy prior to re-innervation. The purpose of this study was to assess the impact of growth hormone (GH) therapy on preventing the deleterious effects of chronic denervation (CD) injury on nerve regeneration and resulting muscle function. We hypothesized that systemic GH therapy can maintain chronically-denervated muscle and SCs, accelerate axonal regeneration, and improve murine extremity function in the setting of chronic denervation (CD) by preserving muscle mass and promoting motor reinnervation.

Abstract 123: Loss Of TGF-beta Activated Kinase (TAK1) Activity Induces Cellular Proliferation And Diminishes Differentiation During Bone Healing

BACKGROUND: Both internal and external distraction devices have been used successfully in correcting midface hypoplasia. Although the indication for surgery and the osteotomy techniques for a Le Fort I and Le Fort III may be similar, deciding when to use an internal vs external device has not been well studied. We studied patient reported outcomes using the FACE Q Patient Reported Outcomes Instrument and functional surveys for internal and external devices for both Le Fort I and Le Fort III patients.