Background: Choice of a biodegradable scaffold and type of stem cells are the major keys to success in tissue engineering in pediatric valve surgery applications. Materials and Methods: This is the in vitro phase of a two-stage tissue engineered stentless pulmonary valve for application in pediatric pulmonary valve surgeries. Effects of in vitro culture conditions, type of stem cells, time and mechanical loading on the retention of mechanical properties of the non-woven melt-blown mesh polymer poly-4-hydroxybutyrate (P4HB), was investigated. Four varying thickness of P4HB were studied to determine the best construct for pulmonary leaflets and its sewing ring. Mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC), alone and in varying combinations, were seeded onto these P4HB scaffolds. A 30-day static and dynamic seeding protocol was followed. All studies were performed in a simulated physiological environment at a temperature of 37 C. Results: P4HB scaffolds of 100 u and 220 u thickness showed an increase uptake of combined MSC and EPC and promoted extracellular matrix formation. P4HB scaffolds seeded with MSC alone showed osteogenic differentiation in all thickness, and little ECM formation. P4HB scaffolds seeded with EPC provided endothelial and interstitial functions and produced ECM on the scaffolds of 100 u and 220 u thickness, but lesser in comparison with that produced by combined MSC-EPC.Conclusions: P4HB scaffolds of 100u and 200u thickness seeded with combined MSC and EPC demonstrated sufficient mechanical properties, which can be further used for in-vivo implantation of a biodegrable stentless pulmonary valve.