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Recreating the Traumatized Muscle Microenvironment: Effects of TGB1 and Topography in Progenitor Cell Culture 1 1 Christopherson GT; 1Ji Y; 2Shin EH; 1Jackson WM; +1,2Nesti LJ Clinical and Experimental Orthopaedics Laboratory, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, 2Department of Orthopaedics and Rehabilitation, Walter Reed National Military Medical Center, Bethesda, MD. [email protected] INTRODUCTION Following orthopaedic trauma, injured muscle tissue becomes populated with mesenchymal progenitor cells (MPCs) containing a cell surface epitope profile and multipotentiality that is characteristic of bone-marrow derived stromal cells (MSCs) [1]. Our laboratory has speculated that these cells are intimately involved in the healing process, playing a key role in wound healing pathologies such as heterotopic ossification (HO). We have evaluated the physical microenvironment of traumatized muscle, and closely recreated the preponderance of nanofiber matrix found in scar tissue with electrospun collagen scaffolds. We hypothesize this microarchitecture helps dysregulate muscle regeneration, working synergistically with inflammatory factors to confuse local MPC populations. This local stem cell confusion enhances fibrosis in lieu of tissue repair, generating an early osteoinductive region, leading to HO. Our specific aims in this study were (1) to evaluate the impact of our biomimetic nanofiber topography on early-passage MPC populations in the presence of a primary inflammatory cytokine, transforming growth factor β1 (TGF-β1) and (2) examine early cellular adhesion to explain how topography can shift baseline osteogenic gene expression and influence phenotypic change. MATERIALS AND METHODS Collagen Nanofibers: Collagen was dissolved in HFIP at 3.5% w/w and electrospun onto grounded coverslips[2]. Small areas of fiber were purposely solvent-melted to produce 2D/fiber interfaces. Samples were glutaraldehyde vapor-saturated for 2 hours, and thoroughly rinsed with PBS for cell culture. Petri dish samples were made in similar fashion. Cell Culture: MPCs were harvested using a previously described protocol [1]. For immunofluorescence study, MPCs (Passage 3 or lower) were seeded onto collagen fiber coverslips and allowed to attach for 5 hours, before phalloidin immunostaining. For RNA isolation, MPCs were seeded in 150-mm nanofiber Petri dishes, with collagen coated Petri dishes as 2D controls (350,000 cells/dish). Cells were cultured for 8 days in media containing 10% FBS with or without 20 ng/mL TGF-β1, with media changes on days 3 and 5. Real Time RT-PCR: RNA was isolated using Trizol phase separation and prepared for use in a 96-well osteogenesis pathway-specific RT2PCR array (SABiosciences). RESULTS A characteristic microscale component of traumatized muscle is randomized fiber matrix, recreated closely by electrospinning (Figure 1). !"#$%!&'()*+$"' %,#)(#%-."/'()*+$"'' "$"+%,0*1)2'+0$$#3"2' %,#)(#%-."/'' 4'56' 4'56' Figure 1: SEM characterization of traumatized tissue. Electrospun collagen closely recreates the physical microenvironment. MPCs cultured on this fiber surface have a significantly different baseline gene expression than cells cultured on 2D: genes primarily involved in fibrosis, ECM and/or cartilage formation are downregulated (higher order collagens 11/12, COL1A1, CDH11, COMP, FN1, FGF2, IGF1, ITGA1). BMP2, a factor highly expressed in early stages of HO formation[3], is upregulated in fiber cultures. In the presence of inflammatory cytokine TGF-β1 additional fibrotic/proliferative genes are downregulated (FGFR2, SOX9), coinciding with an upregulation of genes associated with mineralization and/or HO (BMP2, PHEX), and trophic mediators (CD36, CSF3, CTSK, EGFR). (";<-4,=#&14(' =/-4,=#&14(' "<'+074-6' "<'+074-6' !"#$%&$' !()*' !"#$$&$' +,+-%' 01,&$' 7./%' !6+)' !"./' 01,&%' !"#$2&$' 0,+$' /345' /'8'292:' )>+"#(' !3&<,4' Figure 2: Volcano-plot of osteogenesisspecific PCR array comparing MPCs cultured on fibers and 2D w/TGF-β1 (n=3). Fibrotic genes were downregulated; genes associated with ectopic bone formation and trophic functions were upregulated. Short-term MPC adhesion (1-5h) revealed less-polarized cells that were spread over a larger area following attachment to fibers, as well as a more sharply defined and higher density stress fiber network (Figure 3). !& &'%()**+,-.% '& !"#$% !"#$% !"#$%& "& ()**+,-.%.+.)/01-2% ()**+,-.%.+.)/01-2% Figure 3: MPC short-term adhesion (5 hours). DISCUSSION When cultured on biomimetic collagen fiber matrix, MPCs have a faster and more robust attachment than collagen-coated 2D surfaces. This cytoskeletal change likely triggered a different signaling cascade, contributing to phenotypic shifts. Addition of TGF-β1 further modulates this shift, synergistically decreasing a healthy fibrotic response and upregulating genes associated with bone formation/mineralization and a more trophic cell nature. TGFBR2 – a key mediator of TGF-β1 cell response[4] – is also upregulated on fiber samples, suggesting their cytoskeletal arrangement makes them more sensitive to TGF-β1 induction. SIGNIFICANCE This study helps to define surface topography as a contributing factor to fibrotic cellular function during tissue repair, and emphasizes how preventing firbosis may deprive dysregulated cells of an osteoinductive environment during tissue regeneration and minimize the risk of HO. REFERENCES: 1) Nesti LJ et al. (2008) JBJS Am 90:2390; 2) Li WJ et al. (2005) Biomaterials 26:5158; 3) Tachi K et al. (2011) Tissue Eng A 17:597 4) Huang T et al. (2011) EMBO J 30:1263 ACKNOWLEDGEMENTS: Supported by the Intramural Research Program at the NIH, NIAMS (1ZIAAR041191). Poster No. 1579 • ORS 2012 Annual Meeting