AVBS location:Brisbane, Queensland, Australia date:7-11 September 2006
Most heart valve tissue engineering techniques are based on in vitro seeding of matrix materials. Alternative in vivo seeding of vascular grafts, by means of intraperitoneal (IP) implantation of a non-degradable matrix and induction of a foreign body reaction (FBR) during 2 weeks, has been proposed. We modified this technique using 3 day stimulated endogenous in vivo cell seeding as an alternative for heart valve tissue engineering. First we studied the initial phases (6h, 1, 2, 3, 5,& 7d) of FBR in rats and besides the presence of cells from the immune system we found the presence of stem/progenitor cells in neomatrix tissue around intraperitoneally implanted bovine pericardium matrices. Immunohistochemistry showed Sca-1+ cells immediately after implantation and CD117+ or CD34+ cells 2-3 days after implantation. Lineage negative cells obtained from 3 day implants by magnetic sorting were stained for Sca-1, CD34 and CD117, representing 24.3 ± 9.6; 42.3 ± 9.3 and 63.2 ± 12.8 % of the negative fraction, respectively. These cells also showed several non-erythroid clonal cell aggregates in methylcellulose cultures suitable for stem cell growth.
Next spontaneous endogenous seeding of an acellular crosslinked (photooxidised bovine pericardium) pulmonary valve construct (group 1) was compared to intraperitoneally preseeded constructs (group 2) implanted in sheep. All valves functioned normally before explantation at 1 week (n=6 in each group), 1 month (n=6 in each group) and 5 months (n=8 in each group), except for one thrombosed leaflet in a group 1-valve at 1 month. Macroscopical examination revealed extensive remodelling in the preseeded construct but not in the controls after being in the pulmonary position for 5 months. Significant increased recellularisation was found after IP preseeding compared to spontaneous seeding. Cell coverage was 71 to 100% of the leaflet versus 8 to 26% (p<0.05) respectively, with full coverage in all preseeded valves after 5 months. Tenfold higher neomatrix deposition and doubling of the leaflet thickness was found in group 2 versus group 1 (p<0.05). A concomitant significant decrease in leaflet length (15%) was found at one month in group 2, most probably an early sign of remodelling as observed in valves from the same group after 5 months. The total cross-sectional surface and total amount of collagen (picrosirius red staining) of the original photooxidised pericardium in the leaflet remained unchanged in both groups. Immunohistochemistry showed low immune response (CD45, MHC-1 & 2), stem/progenitor marker expressing cell infiltration (CD34 & CD117), appropriated differentiation (alpha smooth muscle actin and heavy chain myosin), some proliferation (phosphohistone H3) and spontaneous endothelialisation (ecNOS) of the valves.
In conclusions, short term IP preseeding provides a combination of cell types including stem/progenitor cells allowing a heart valve graft to remodel into a recellularised and functioning heart valve. These valves contained myofibroblasts early after implantation and showed spontaneous endothelialisation with a full monolayer after 5 months.