Introduction: The incorporation of carbon monoxide (CO) has the potential to improve patency of tissue engineered vascular grafts since CO can enhance endothelialization in appropriate doses[1]. Further, endogenous levels of CO have recently been shown to be essential in cell signaling. For therapeutic applications, controlling the CO dose is important both for a current clinical trial to treat pulmonary fibrosis and for potential vascular applications. Our goal is to provide controlled, local delivery of CO by incorporating visible light activated carbon monoxide releasing molecules (CORM) within electrospun scaffolds that can provide a hydrophobic barrier needed to allow CORM activation. The goals of this study were to determine CORM loading concentrations and scaffold compositions that enable extended CO releases profiles in cell culture conditions as well as vascular cell attachment and function.
Materials and Methods: PCL and 75% PCL/collagen solutions were electrospun with incorporated CORMs (0, 2, and 4% w/w). Fiber diameters and collagen content were characterized with SEM and EDS, respectively. CORMs / Scaffolds were incubated and activated in cell culture conditions (DMEM, 10% FBS) using 470 nm light. CO release was confirmed directly with a myoglobin assay and indirectly with fluorescence at 350/450 nm[2]. For release profiles, the fluorescent intensity was determined for different activation times (up to 60 min) and incubation times (up to 3 days). Rat smooth muscle cells (SMC) and primary rat aortic endothelial cells were seeded on the scaffolds to test cell viability (DNA assay) and phenotype.
Result and Discussion: Solution concentrations were adjusted to obtain similar average fiber diameters for PCL scaffolds with 0 and 2% CORM loading (1.3±0.4 and 1.5±0.1, respectively). Fluorescence intensity, indicating CO release, was greatest after 30 min of activation in cell culture conditions. CO release from PCL meshes occurred after up to 60 min of pre-incubation in cell culture conditions. The myoglobin assay confirmed a high CO yield for scaffolds (e.g., 92% for PCL with 2% w/w CORM). Neither the CORM itself nor the released CO were toxic SMCs at 2% loading (n=9, 1-way ANOVA, Tukey). However, the cellular response to activation was limited. Thus, we are including higher CORM loadings (i.e., 4%) as well as collagen to modulate the endothelial cell response. In preliminary work, we demonstrated that CORM in collagen/PCL meshes can still be activated after 60 min of pre-incubation.
Conclusion: The results show that the release of CO from CORM-loaded electrospun meshes can be controlled by varying irradiation time. While the CORM was not toxic at 2% loading, the early results suggest that a higher dose of CO is required to impact vascular cell function. The addition of collagen within the electrospun mesh provided cell-binding sequences and still allowed for activation after similar pre-incubation times demonstrated with pure PCL.
Support from The National Science Foundation
References:
[1] Sato K, Balla J, Otterbein L, Smith RN, Brouard S, Lin Y, et al. Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants. J Immunol 2001;166:4185–94. doi:10.4049/jimmunol.166.6.4185.
[2] Peng P, Wang C, Shi Z, Johns VK, Ma L, Oyer J, et al. Visible-light activatable organic CO-releasing molecules (PhotoCORMs) that simultaneously generate fluorophores. Org Biomol Chem 2013;11:6671–4. doi:10.1039/c3ob41385c.