Tissue Engineering of Ovine Aortic Blood Vessel Substitutes Using Applied Shear Stress and Enzymatically Derived Vascular Smooth Muscle Cells

Abstract

Compared to native blood vessels, all clinically available blood vessel substitutes perform suboptimally. Numerous approaches to tissue engineer (TE) blood vessels have been pursued using different scaffold materials, cell types, and culture conditions. Several limitations however remain to be overcome prior to the potential application in the arterial system. This study aimed at tissue engineering viable ovine blood vessels suitable for implantation into the systemic circulation of sheep. In recent studies vascular smooth muscle cells (vSMC) were derived by an explant technique. However, in this study we show that homogenous populations of differentiated vSMC were only obtained by enzymatic dispersion as characterized by immunostaining for specific vSMC marker proteins. In contrast the explant method yielded predominantly less differentiated myofibroblast-like cells. Enzymatically derived vSMC were seeded onto P-4-HB scaffolds and incubated either in a pulsatile flow bioreactor or under static conditions. Dynamically cultured TE blood vessel substitutes showed confluent layered tissue formation and were completely water resistant. They displayed significantly increased ECM synthesis, DNA, and protein content as well as vSMC marker expression. Mechanical properties of bioreactor cultured TE blood vessels approached those of native aorta. In conclusion ovine, aortic blood vessel substitutes were successfully created using enzymatically derived vSMC, bioabsorbable scaffolds, and applied shear stress.

Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering

“…a) Scanning electron microscopy-For ultrastructural analysis, unseeded and seeded scaffold samples were processed for characterization by scanning electron microscopy (SEM) as described previously [36]. Fiber samples were cut from different locations on the electrospun mat to obtain representative fibers.…”

“…The transmission electron microscope was operated at 100 kV. c) Multiphoton imaging and SHG microscopy-To determine the fiber composition in the electrospun scaffolds, multiphoton imaging and SHG microscopy were performed using a Zeiss LSM 510 META NLO femtosecond laser scanning system (Carl Zeiss MicroImaging Inc., Thornwood, NY, USA), coupled to a software-tunable Coherent Chameleon titanium:sapphire laser (720 nm -930 nm, 90 MHz; Coherent Laser Group, Santa Clara, CA, USA), and equipped with a high-resolution AxioCam HRc camera with 1300 × 1030 pixels (Carl Zeiss) as previously described [36][37][38]. Images were collected using an oil immersion Plan-Neofluar 40×/1.3 numerical aperture (NA) DIC, or an oil immersion Plan Apochromat 63×/1.4 NA objective lens (Carl Zeiss).…”

“…All observations were made using non-treated samples of collagen10%/elastin5% (not cross-linked, cross-linked, and with PCL10%) and gelatin10% (not cross-linked, cross-linked, and with PCL10%) scaffolds. ECM structure-dependent autofluorescence and SHG were induced using wavelengths of 760 nm (elastin) and 840 nm (collagen) as described previously in more detail [36][37][38]. Non-invasive serial optical horizontal sections of six different areas of each of the specimens were taken in z-steps of 1 μm, 2.5 μm or 5 μm to depths of 50-100 μm.…”

Biodegradable elastomeric scaffolds with basic fibroblast growth factor release

“…This should be the case for both tissues that will be dynamically cultured in vitro, or where constructs are placed for development in situ. [1][2][3][4] A number of research groups have thus been interested in developing biodegradable polymers and scaffolds with mechanical properties appropriate for application in the engineering of soft tissues. [5][6][7][8] In addition to appropriate mechanical properties other biofunctionality would be attractive for such scaffolds, for instance the controlled release of growth factors to stimulate local angiogenesis toward the developing tissue.…”

The Future of Cell Therapy and Tissue Engineering in Cardiovascular Disease: The New Era of Biological Therapeutics

“…Indeed, SM--actin has been used as a marker of early SMC differentiation, followed by calponin, h-caldesmon and SM myosin (Owens GK 1995;Hungerford JE 1996;Katoh Y 1996;Thyberg J 1996;Hungerford JE 1999). It is well known that SMCs grown in vitro can undergo a phenotypic cell transition from the normally quiescent 'contractile' status observed in vivo to a proliferative-secretory state (Thyberg J 1996;Opitz F 2004 myosin variants MyHC-A pla1, MyHC-A, and MyHC-B (Somlyo AP 1993). These tendencies depend, however, on cellular density, serum concentration, and substrate for attachment.…”