• Home
  • Search Results
  • Generating elastin-rich small intestinal submucosa-based smooth muscle constructs utilizing exogenous growth factors and cyclic mechanical stimulation.

Generating elastin-rich small intestinal submucosa-based smooth muscle constructs utilizing exogenous growth factors and cyclic mechanical stimulation.

Tissue engineering. Part A (2009-07-03)
Rebecca Long Heise, Julia Ivanova, Aron Parekh, Michael S Sacks
ABSTRACT

Successful approaches to tissue engineering smooth muscle tissues utilize biodegradable scaffolds seeded with autologous cells. One common problem in using biological scaffolds specifically is the difficulty of inducing cellular penetration and controlling de novo extracellular matrix deposition/remodeling in vitro. Our hypothesis was that small intestinal submucosa (SIS) exposed to specific mechanical stimulation regimes would modulate the synthesis of de novo collagen and elastin by bladder smooth muscle cells (BSMC) within the SIS matrix. We further hypothesized that the cytokines vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), two key growth factors involved in epithelial mesenchymal signaling, will promote the cellular penetration into SIS necessary for mechanical stimulation. BSMC were seeded at 0.5 x 10(6) cells/cm(2) onto the luminal side of SIS specimens. VEGF (10 ng/mL) and FGF-2 (5 ng/mL) were added to each insert in the media every other day for up to 7 days in static culture. Following static culture, specimens were stretched strip-biaxially under 15% peak strain at either 0.5 or 0.1 Hz for an additional 7 days. Following the culture period, specimens were assayed histologically and biochemically for cellular penetration, proliferation, elastin, collagen, and protease activity. Histological analyses demonstrated that in standard culture media, BSMC remained on the surface of the SIS while both FGF-2 and VEGF profoundly promoted ingrowth of the BSMC into the SIS. The penetration of the cells in response to these cytokines was confirmed using a Transwell assay. Following cellular penetration, BSMC produced significant amounts of elastic fibers under cyclic mechanical stretching at 0.1 Hz under 15% stretch, as evidenced by colorimetric assay and histology using a Verhoeff-Van Gieson stain. Protease activity was assessed in the media and found to be statistically increased in static culture following FGF-2 treatment. These findings demonstrate, for the first time, the capability of BSMC to produce histologically apparent elastin fibers in vitro. Moreover, our results suggest that a strategy involving growth factors and controlled mechanical stimulation may be used to engineer functional, elastin-rich tissue replacements using decellularized biologically derived scaffolds.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Acetic acid, glacial, ReagentPlus®, ≥99%
Sigma-Aldrich
Acetic acid, glacial, ACS reagent, ≥99.7%
Sigma-Aldrich
Acetic acid, glacial, puriss., meets analytical specification of Ph. Eur., BP, USP, FCC, 99.8-100.5%
Sigma-Aldrich
Acetic acid, glacial, ≥99.99% trace metals basis
Sigma-Aldrich
Acetic acid, natural, ≥99.5%, FG
Supelco
Acetic acid, analytical standard
Sigma-Aldrich
Acetic acid, glacial, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.8%
Sigma-Aldrich
Acetic acid, ≥99.5%, FCC, FG
Sigma-Aldrich
Acetic acid, for luminescence, BioUltra, ≥99.5% (GC)
Sigma-Aldrich
Acetic acid, glacial, puriss., 99-100%
Sigma-Aldrich
PureCol, protein from bovine hide, suitable for cell culture