Introduction: The development of tissue engineered vascular grafts (TEVGs) requires large quantities of uniformly distributed vascular smooth muscle cells (VSMCs) in biomaterial scaffolds, VSMC proliferation, and induction of extracellular matrix (ECM) production[1]. A biodegradable polyurethane scaffold (D-PHI) has been shown to support an anti-inflammatory phenotype for monocytes, which are one of the first cells to migrate to an implant site and provide vital signalling for wound healing and new tissue regeneration[2]. Vitamin C (Vc) derivatives ( ascorbic acid (AA) and sodium ascorbate (SA)), have been shown to stimulate VSMC growth and ECM production, and modulate cytokine-release from monocytes[3]-[6]. The objective of the current study was to investigate cell seeding strategies on D-PHI, and to use the identified conditions to compare AA and SA with respect to supporting ECM production and maintaining a desired VSMC phenotype.
Materials and Methods: D-PHI scaffolds were prepared using a previously established protocol[7]. Human coronary artery SMC (Lonza CC-2583) seeding optimization was performed on D-PHI disk-formed scaffolds (6 mm diameter, 1 mm thickness) to find the maximum number of cells that could be seeded as a function of time, frequency of seeding, and the seeding volume. Seeded scaffolds were analyzed for total DNA and stained by hematoxylin and eosin (H&E) for histology. 300,000 (300k) VSMCs were seeded onto each disk scaffold and treated with 50 µM, 100µM or 200µM AA or SA for 1 week and characterized for DNA and ECM production (glycosaminoglycans (GAG), collagen and elastin). Data were analyzed by analysis of variance (SPSS 17.0). Statistical significance was reported for p < 0.05.
Results and Discussion: The highest scaffold seeding efficiency was achieved with 75k or 150k VSMCs (Fig 1A). However, scaffolds seeded with 75k up to 300k VSMCs all demonstrated good cell infiltration into the material (Fig 1B). 300k VSMCs was selected to be used to study the optimal loading volume despite having lower seeding efficiency since it achieved the greatest number of loaded cells with good infiltration. Of the loading volumes tested, 8.3 µl allowed maximum cell uptake into the porous scaffolds (Fig 1C). Given that lower cell concentrations seeded with greater efficiency, a two-stage seeding protocol with 150k cells/stage, loaded over 48 hr using the optimal volume of 8.3µl, was assessed (Fig. 1). The final seeding efficiency of this latter approach was close to 85% (Fig 1D).
AA and SA comparative data are reported in Fig. 2 and show that SA outperformed AA in inducing GAG (50 µM, 100µM and 200µM) and elastin (100µM and 200µM) production.
Conclusion: This study has identified important seeding conditions that improved cellularity in D-PHI scaffolds. When used to compare AA vs SA on DNA and ECM protein production it was found that SA outperformed AA. These conditions will be used for future TEVG development in co-culture with human monocytes.
Canadian Institutes of Health Research operating grant #230762.
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