Background: Calcific aortic valve disease (CAVD) is a frequent cardiac pathology in the aging society. Although valvular interstitial cells (VIC) seem to play a crucial role, mechanisms of CAVD are not fully understood. Development of tissue-engineered cellular models by 3D-bioprinting may help to further investigate underlying mechanisms of CAVD. Methods: VIC were isolated from ovine aortic valves and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM). VIC of passages six to ten were dissolved in a hydrogel consisting of 2% alginate and 8% gelatin with a concentration of 2x106 VIC/ml. Cell-free and VIC-laden hydrogels were printed with an extrusion-based 3D-bioprinter (3D-Bioplotter® Developer Series, EnvisionTec, Gladbeck, Germany), cross-linked and incubated for up to 28 days. Accuracy and durability of scaffolds was examined by microscopy and cell viability was tested by cell counting kit-8 assay and live/dead staining. Results: 3D-bioprinting of scaffolds was most accurate with a printing pressure of P<400 hPa, nozzle speed of v<20 mm/s, hydrogel temperature of TH=37 °C and platform temperature of TP=5 °C in a 90° parallel line as well as in a honeycomb pattern. Dissolving the hydrogel components in DMEM increased VIC viability on day 21 by 2.5-fold compared to regular 0.5% saline-based hydrogels (p<0.01). Examination at day 7 revealed dividing and proliferating cells. After 21 days the entire printed scaffolds were filled with proliferating cells. Live/dead cell viability/cytotoxicity staining confirmed beneficial effects of DMEM-based cell-laden VIC hydrogel scaffolds even 28 days after printing. Conclusions: By using low pressure printing methods, we were able to successfully culture cell-laden 3D-bioprinted VIC scaffolds for up to 28 days. Using DMEM-based hydrogels can significantly improve the long-term cell viability and overcome printing-related cell damage. Therefore, future applications 3D-bioprinting of VIC might enable the development of novel tissue engineered cellular 3D-models to examine mechanisms involved in initiation and progression of CAVD.

No abstract

No abstract