Pulsatile Perfusion Of Cardiovascular Progenitor Cells Drives Vascular Smooth Muscle Differentiation And A Contractile Phenotype
Timothy P. Martens, MD, PhD, Cole J. Knox, MS, Sean Wilson, PhD, Mary Kearns-Jonker, PhD, Anees J. Razzouk, MD.
Loma Linda University, Loma Linda, CA, USA.
Objective(s): Ex vivo maturation of tissue engineered constructs (ECs) may yield implants capable of somatic growth. We employed a pulsatile perfusion system to drive differentiation of cardiovascular progenitor cells (CPCs) seeded on decellularized pulmonary artery down a vascular smooth muscle lineage with a contractile phenotype.
Methods: 5 million CPCs were seeded by intramural injection and the constructs placed in static or pulsatile perfusion culture for 1 or 3 weeks. ECs were evaluated by immunohistochemistry and PCR. Mechanical properties of seeded constructs were compared to native tissue and decellularized controls. ECs were implanted into juvenile lambs for 14 days.
Results: Decellularization removed 99.1+/-0.3% of native DNA. Histology of decellularized constructs revealed preservation of collagen and elastin fibers. Static culture of 5 million CPCs demonstrated retention of cultured CPCs at 1 week (2.0+/-0.3x10^6 CPCs, n=6) and proliferation at 2 weeks (6.5+/-0.9x10^6 CPCs, n=4). Three weeks of pulsatile culture but not static culture was associated with upregulation of MYH11 and CNN, both markers of contractile smooth muscle. Three weeks of pulsatile culture generated constructs capable of radial contraction similar to native tissue (2.3+/-0.4 vs. 2.8+/-0.5 grams, EC vs. native, n=6) whereas all other culture conditions yielded tension similar to unseeded controls (1.5 +/- 0.2 grams, p < 0.05, n=6). ECs implanted in 3 month old lambs (n=3) generated no adverse events over two weeks of observation.
Conclusions: Ex vivo maturation of CPCs under physiologic conditions generates engineered constructs with intrinsic contractility. Long term implant studies are needed to assess potential for somatic growth.