oa Influence of Scaffold Structure on Cell Behaviour for Valve Tissue Engineering

The success of a tissue engineered heart valve is dependent on developing the right structure, the right interactions between the cells and the right matrix and mechanical force. Different materials have been used as scaffold, however the best processing method have not been established. We have used 2 different type of scaffold: collagen based scaffold and a nanofibrillar scaffold made from poly(ε-caprolactone) (PCL). Different seeding methods have been tested for cell compatibility of these scaffolds with human adipose derived mesenchymal stem cells (hADSCs) and with human telomerase immortalized bone marrow derived stem cells (hTERT). Nanofibrillar scaffolds have been produced by jet-spraying the polymer on a metal grid. Collagen scaffolds were made by freeze drying a 1% bovine collagen solution chemically crosslinked with EDC-NHS. Morphological evaluations of the structures were performed using scanning electron microscopy. Elastic modulus of dry scaffolds (10x6x1 mm, n=5) was measured with a planar biaxial test bench with displacement rate of 0.05 mm/s. Two different type of cells (600000/scaffolds) were seeded using different seeding methods to find the best condition: top seeding for 2 hours and then dynamically cultured using a rotary mixer, directly dynamically seeded for different time period and with different volume (5, 10 and 25 ml). DNA quantification using Hoescht 3258 and DAPI staining were used to evaluate proliferation and penetration inside the scaffold. Immunohistochemistry was used to check collagen production. PCL scaffolds were composed of non woven nanofibers (600 nm average diameter) assembled in a highly open structure. Collagen scaffold showed an interconnected porous structure with average pore size of 100 um. Nanofibrillar scaffolds showed higher elastic modulus compared to collagen (200 KPa compared to 150 KPa). The different cell seeding approaches had an effect on cellular distribution and cell number. With 10 ml of volume cells attached more after 24 hours compared to 5 ml with no further difference compared to 25 ml. Top-seeded matrices resulted in a high cell concentration on the seeded surface while rotary seeding allowed cells to attach on both scaffold sides but in fewer numbers. Regardless of seeding method, cells proliferated extensively (up to 10 and 2-fold DNA increase for hTERT and hADSC respectively ) on both scaffold, but proliferation was up to twice higher within nanofibrillar structures compared to collagen scaffolds. Both cell types were able to populate the entire area of both scaffolds over 10 and 14 culture days for hADSC and hTERT respectively. Both cell type produced their own ECM within the scaffolds as indicated by collagen I positive staining. Jet-sprayed PCL nanofibrillar scaffolds are a promising alternative to collagen scaffolds for cellular infiltration and proliferation.