A major issue confronting tissue engineering (TE) is the poor survival of implanted cells in vivo, due tohypoxia and loss of cell/cell attachment. Techniques that diminish the influence of these factors were investigated in rat and mouse TE models, where a vascular pedicle is enclosed in a plastic TE chamber in vivo. The vascular pedicle sprouts capillaries within a week, but a hypoxic environment exists for 10 days. Our group has investigated three techniques to significantly improve implanted cell survival in vivo. Methods and Results: In minimizing hypoxic cell death two techniques have been used: 1) A delayed implantation/prevascularized rat chamber technique in which rat myoblasts were implanted at either 0, 4 or 7 days post pedicle/chamber construction. Capillaries increase in this model from zero at day 0 to an extensive capillary network at day 14. DiI labeled myoblasts/mm2 survival (counted morphometrically) 7 days post implantation was positively correlated to the increase in vascularization within the chamber (p=0.017). 2) In vitro preconditioning of L6 myoblasts with a number of ‘preconditioning’ agents including heat, or hypoxia, or DETA-NONOate (nitric oxide donor) demonstrated that preconditioning with DETA-NONOate in vitro prior to in vivo implantation significantly increased percent myoblast volume (p=0.007) after 3 weeks in SCID mouse TE chambers. Phosphoimmunoblot analysis of pro-survival signaling pathways revealed significant activation of serine threonine kinase Akt (p < 0.01) and ERK 1/2 (p < 0.05) signaling with DETA-NONOate preconditioning. 3)To counteract loss of adjacent cell attachments causing implanted cell death, the survival of liver progenitor cell (LPC) spheroids (22,500 cells/spheroid) compared to the same number of single LPCs in the mouse TE chamber demonstrated spheroid implantation significantly increased LPC survival (p<0.05) and differentiation (p<0.005).
Conclusion: Prevascularization, cell preconditioning, and multicellular-spheroid implantation offer significant potential to improve implanted cell survival and tissue engineering outcomes.