OTA 2013 Posters

Scientific Poster #124 Basic Science OTA 2013

An Innovative Culture System for the Study of Heterotopic Ossification

Husain Bharmal, MD1,2; Gregory T. Christopherson, PhD1; Youngmi Ji, PhD1; Carl Cirino1; Wesley M. Jackson, PhD3; Leon J. Nesti, MD, PhD1,2,3;
1Orthopaedic Research Group, National Institute of Arthritis and Musculoskeletal and
Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA;
2Department of Orthopaedics and Rehabilitation, Walter Reed National Military Medical Center, Bethesda, Maryland, USA;
3Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA

Background/Purpose: Following orthopaedic trauma, injured muscle tissue becomes populated with mesenchymal progenitor cells (MPCs) hypothesized to play a key role in wound healing pathologies such as heterotopic ossification (HO). During recent military operations, there has been a significant increase in the prevalence of heterotopic ossification in the combat-wounded patient population with rates as high 64%. We hypothesize the physical microstructure (fibrosis) within a wound dysregulates muscle regeneration, working synergistically with inflammatory factors to confuse local MPC populations and generate an early osteoinductive region. In this study, we examine the structure of decellularized early-stage traumatized muscle tissue taken from patients who develop HO and identify noncellular structural components that may be instrumental in pathological wound development and create a biomimetic in vitro culture platform replicating the wound microarchitecture to create an in vitro model system for studying the development of HO.

Methods: Tissue decellularization: Surgical waste muscle tissue was obtained from consented patients injured during Operation Enduring Freedom; these patients were radiographically observed as developing HO in subsequent months. Tissue was sectioned into 5-mm3 segments and incubated overnight in 0.1% sodium dodecyl sulfate (SDS) surfactant solution to remove all cellular and lipid components characterized using scanning electron microscopy (SEM) and replicated the microstructure by electrospinning collagen. Cell isolation and culture: MPCs were harvested from traumatized muscle by plastic-adherence methodology and expanded on collagen-coated and fiber matrix dishes for 3 weeks, before ensuing culture in osteogenic media (Invitrogen) for 2 weeks. Calcium deposition was measured with alizarin red, and osteoblast activity was assayed with alkaline phosphatase. Protein lysates were also collected for Western blotting analysis.

Results: We have evaluated the physical microenvironment of traumatized muscle tissue in patients radiographically confirmed to form HO by lysing all cellular components from the tissue, leaving a preponderance of nanofiber matrix. This nanofiber component was identically replicated with electrospun collagen scaffolds. MPCs cultured on this fiber surface have a significantly increased osteogenic potential, with greatly increased calcium deposition (as measured by alizarin red) and alkaline phosphatase expression compared to cells cultured on 2-dimensional collagen. Western blotting analysis revealed increased vimentin expression for MPCs cultured on fiber substrates prior to osteogenesis, as well as a decrease in alpha smooth muscle actin (αSMA), a prominent fibroblast marker. Vimentin has been shown to be essential for cartilage formation, which is hypothesized to precede endochondral ossification of HO. It has also been shown that decreases in vimentin correlate with osteoblast differentiation and bone formation, perhaps acting as a trigger for osteocalcin signaling.

Conclusion: Our experimental results show a robust increase in calcium secretion from MPCs cultured on a biomimetic nanofiber matrix. The increase in vimentin expression before osteogenic induction correlates well with literature, with a corresponding decrease in fibrotic markers (αSMA) indicating a shift into preosteogenic state. The sensitivity of osteogenic phenotype to the nanofiber matrix suggests that it is likely a key component for properly elucidating HO development using in vitro cellular models.

Alphabetical Disclosure Listing

• The FDA has not cleared this drug and/or medical device for the use described in this presentation   (i.e., the drug or medical device is being discussed for an “off label” use).  ◆FDA information not available at time of printing. Δ OTA Grant.