Download Recreating the Traumatized Muscle Microenvironment

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).
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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).
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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).
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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