Generating Elastin-Rich Small Intestinal Submucosa–Based Smooth Muscle Constructs Utilizing Exogenous Growth Factors and Cyclic Mechanical Stimulation

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 0.5Â10 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 stripbiaxially 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.

Optical assessment of tissue anisotropy in <italic>ex vivo</italic> distended rat bladders

“…For example, Helse et al, have shown that mechanical stimulation of the in vitro tissue engineered bladders triggers elastin production and improves bladder compliance. 9 Knowing the regions of the bladder which possess high content of elastin (e.g., dorsal zone according to our results) and their organization, tissue engineers can apply the optimum mechanical stimulation. In addition, our results demonstrate that the different regions of ex vivo distended bladders exhibit very different (thickness independent) anisotropy.…”

“…One promising solution is replacing the bladder with engineered tissues. 8,9 A major outstanding issue in this approach is ensuring comparable functionality through comparable engineered architecture, required for a healthy normal bladder. Thus, currently ambiguous relationships between distension-induced changes in the organization of smooth muscle and extracellular matrix tissues that comprise the bladder wall, and the resulting alterations in bladder function, warrant further investigation.…”

Leveraging “Raw Materials” as Building Blocks and Bioactive Signals in Regenerative Medicine

“…132,135 In addition to these native ECM molecules, the collagen fiber orientation that is maintained after the decell process has also attracted attention. 132 Both of these inherent properties have sparked strategies employing SIS as scaffolds in the fields of cardiovascular, [137][138][139][140] bone, [141][142][143] nerve, 144 soft tissue, 86,87,145 and urogenital [146][147][148][149] tissue engineering (Table 6). Currently, SIS is Food and Drug Administration (FDA) approved for several urogenital applications, including hernia repair.…”

Tissue Engineering for Tissue and Organ Regeneration

“…(Kohn, 2002) Cyclical mechanical forces have also been demonstrated necessary for bladder smooth muscle growth and development while static and non-contractile environments are less likely to produce robust contractile responses. (Heise, 2009) POC [poly (1,8-octanediol-cocitrate)] is a relatively newly described member of a family of diols which is a synthetic polymer that has a wide range of applications including those for very elastic tissues such blood vessels, orthopedic applications, and perivascular wraps. Qiu, 2006;Serrano, 2011) The elastic potential of this polymer is dependent upon several polymerization properties including the length and temperature of scaffold crosslinking.…”