Eboda Oluwatobi

Diminished Chondrogenesis and Enhanced Osteoclastogenesis in Leptin-Deficient Diabetic Mice (ob/ob) Impair Pathologic, Trauma-Induced Heterotopic Ossification

Diabetic trauma patients exhibit delayed postsurgical wound, bony healing, and dysregulated bone development. However, the impact of diabetes on the pathologic development of ectopic bone or heterotopic ossification (HO) following trauma is unknown. In this study, we use leptin-deficient mice as a model for type 2 diabetes to understand how post-traumatic HO development may be affected by this disease process. Male leptin-deficient (ob/ob) or wild-type (C57BL/6 background) mice aged 6-8 weeks underwent 30% total body surface area burn injury with left hind limb Achilles tenotomy. Micro-CT (μCT) imaging showed significantly lower HO volumes in diabetic mice compared with wild-type controls (0.70 vs. 7.02 mm(3), P < 0.01) 9 weeks after trauma. Ob/ob mice showed evidence of HO resorption between weeks 5 and 9. Quantitative real time PCR (qRT-PCR) demonstrated high Vegfa levels in ob/ob mice, which was followed by disorganized vessel growth at 7 weeks. We noted diminished chondrogenic gene expression (SOX9) and diminished cartilage formation at 5 days and 3 weeks, respectively. Tartrate-resistant acid phosphatase stain showed increased osteoclast presence in normal native bone and pathologic ectopic bone in ob/ob mice. Our findings suggest that early diminished HO in ob/ob mice is related to diminished chondrogenic differentiation, while later bone resorption is related to osteoclast presence.

Role of Anatomical Region and Hypoxia on Angiogenic Markers in Adipose-Derived Stromal Cells

BACKGROUND Recent research into adipose-derived stem cells (ASCs) suggests that anatomical location has a major impact on the metabolic profile and differentiation capacity of ASCs. By having a better understanding of how various ASCs respond to cellular stressors such as hypoxia, which are induced during routine surgical procedures, we can facilitate future development of cell-based therapies to improve wound healing. PATIENTS AND METHODS Human ASCs were isolated from the superficial and deep adipose layers of four patients undergoing elective abdominoplasty. ASCs were cultured in hypoxic (1% O2, 5% CO2, and 94% N2) conditions. After 12 and 48 hours, ASCs were assessed for markers of angiogenesis by mRNA levels of vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B), and hypoxia inducible factor 1 α (HIF-1α). Western blot analysis was performed to assess levels of VEGF-A, p-NF-κB, and NF-κB. In addition, in vitro analysis of angiogenesis was performed using Matrigel assay (BD Biosciences, Franklin Lakes, NJ). RESULTS We observed significant increases in deep ASCs VEGF-A, VEGF-B, and HIF-1α mRNA expression compared with the superficial layer after 24-hour hypoxia (p < 0.05). Similar results were found when examining protein expression levels, with the deep ASCs expressing significantly larger amounts of VEGF-A and p-NF-κB (p < 0.05) compared with the superficial layer. CONCLUSION Our results suggest that significant variations exist in the angiogenic profile of superficial and deep ASCs. We demonstrate that superficial ASCs are less prone to transcribe potent chemokines for angiogenesis, such as VEGF-A, VEGF-B, and HIF-1α and are less likely to translate VEGF-A and NF-κB. This may help with the selection of specific stem cell donor sites in future models for stem cell therapy.

Abstract 89: Novel Targeting of the Alk-2 Receptor Using the Cre/lox System to Enhance Osseous Regeneration by Mesenchymal Stem Cells.

PurPose: There is a significant need for readily available autogenous tissue to aid in bone regeneration without causing a donor-site defect. Most studies focus on the ability of bone morphogenetic protein ligands (BMP-2 and 7) to stimulate the Bmpr1b receptor (ALK-6). The ACVR1 gene is often overlooked, but provides instructions for making the activin receptor type I protein (ALK-2), a protein member of the Bmpr1 family. When the ALK-2 receptor is overexpressed globally, patients develop fibrodysplasia ossificans progressive. We believe this highly osteogenic phenotype can be harnessed in adipose derived stem cells (ASCs) to improve bone tissue engineering. Our goal here was to demonstrate that ALK-2 may serve as a novel target to 1) improve in vitro ASC osteogenic differentiation and 2) enhance in vivo bone regeneration and calvarial healing.

The systemic effect of burn injury and trauma on muscle and bone mass and composition

Background: By understanding the global inflammatory effects on distant myopathies, surgeons can better guide the rehabilitative process for burn patients. The authors tested the systemic effect of burn injury on distant injured muscle and native bone using immunohistochemistry and validated a new morphometric analytic modality to reproducibly quantify muscle atrophy using computed tomographic imaging. Methods: In vivo studies were performed on C57/BL6 mice using an Achilles tenotomy with concurrent burn injury model. Total muscle and bone (tibia and fibula) volume/density were quantified near the site of Achilles tenotomy using micro–computed tomography at 5, 7, and 9 to 12 weeks after surgery. The impact of burn injury on the inflammatory cascade [nuclear factor (NF)-&kgr;B, p-NF-&kgr;B] and the interconnected protein catabolism signaling pathway (Atrogin-1) was assessed by immunohistochemistry. Results: Muscle volume and density at the site of Achilles tenotomy in burned mice were significantly diminished compared with nonburned mice at 5 weeks and 9 to 12 weeks. Similar decreases in muscle volume and density were observed when comparing tenotomy to no tenotomy. Cortical bone health remained stable in burn/tenotomy mice compared with tenotomy. Muscle atrophy was associated with up-regulation of p-NF-&kgr;B, NF-&kgr;B, and Atrogin-1 assessed by immunohistochemistry. Conclusions: Burn injury significantly decreases muscle volume and density. Increased muscle atrophy using our computed tomographic morphometric analysis correlated with a significant increase in intramuscular inflammatory markers and proteolysis enzymes. This study demonstrates a unique characterization of how burn injuries may worsen local myopathy. Moreover, it provides a novel approach for quantifying muscle atrophy over an expanded period.

Abstract 156: Optimizing Timing and Targeting of Bone Morphogenetic Protein Receptor One to Treat Heterotopic Ossification with Novel Kinase Inhibitors and Ligand Traps.

www.PRSJournal.com 111 Sarday, M ay 16 RESULTS: It was found that, relative to the normoxic baseline, hypoxia significantly affected the expression of a range of angiogenesis-related factors including VEGF, ANG and TSP-1. HPP obtained after 5 days culture had a five-fold higher concentration of the angiogenesis-stimulator VEGF, and a two-fold lower concentration of the angionesis-inhibitor TSP-1, compared to fresh plasma (p<0.05). While both factors eventually underwent down-regulation over time under hypoxia, there was significant variation in their temporal expression profile. Releasates of HPP factor-loaded gels increased endothelial cell tubule formation and directional migration relative to control medium, while microvessel sprouting was found to be significantly improved compared to recombinant VEGF (p<0.05). Two patients with non-healing wounds treated with application of 5% HPP factor-containing emulsion showed complete wound closure within 20 and 50 days, respectively.

Abstract 20: Defining the Role of the Vasculogenic Niche in Trauma-induced Heterotopic Ossification using Novel Imaging and Lineage-tracing Mice.

PURPOSE: Heterotopic ossification (HO) is extra-skeletal lamellar bone in soft tissues following trauma. Prior to HO deposition, patients develop inflammation and hypervascularization. Endothelial progenitor cells contribute to neo-angiogenesis by differentiating into mature endothelium. We hypothesized that angiogenesis establishes the niche necessary for HO, and that endothelial cells contribute to HO thereby providing a target for future inhibition.

Abstract 148: use of bmp type I alk-3 knockout mice to develop novel treatment strategies for trauma-induced heterotopic ossification.

PURPOSE: Heterotopic ossification (HO) is a debilitating process characterized by the formation of ectopic bone following trauma, burn injury, and amputations. HO formation in trauma and burn patients is poorly understood at a mechanistic level, contributing to the lack of therapeutic options. In this study, we demonstrate the role of BMP type I receptor Alk3 in HO development and target downstream Alk3 signaling with a novel small molecule (LDN-193189) to prevent HO.