(42h) Engineering Microcapillaries Using Chitosan-Gelatin Injectable Hydrogel Formulations

Engineering Microcapillaries Using Chitosan-Gelatin Injectable Hydrogel Formulations

Blood circulation, waste removal, and exchange of oxygen and nutrients to the human body takes place through complex circulatory system containing various sizes of microvessels such as arteries, capallaries, the heart, and veins.  The attractive feature of microvessels is that they have the ability to grow and remodel in response to the tissue requirement.  Human Umbilical Vein Endothelial Cells (HUVECs) are known to readily self-assemble into tubular structures and ECs are sensors that responds to diverse signals generated in the blood, subendothelium, and interacting cells.  Creation microvessels with different sizes are a challenge as the previous approaches failed to gain control over size during microvascularization.  To address the size controlled microvasculature we proposed to use simple printing of hydrogels containing cells with different needle sizes.  We questioned whether we could use this technology to form different sizes of blood vessels.  Chitosan–gelatin solution is known to facilitate colonization of HUVECs; therefore hydrogels were synthesized with chitosan–gelatin.  Hydrogels mixed with cells were printed by varying factors such as cell density, and needle size and type; and this study gave feasible solution to form viable and size controlled blood vessels. 

Hydrogels were prepared with different combinations of chitosan-gelatin in presence of 2-glycerol phosphate in sterile condition.  Needle size, and end type were investigated for changes in fiber size.  Human Umbilical Vein Endothelial Cells (HUVEC-2) derived from single donors (BD Biosciences, San Jose, CA) were cultured in Medium 200 PRF, with 2% v/v fetal bovine serum, 1 µg/mL hydrocortisone, 10 ng/mL human epidermal growth factor, 3 ng/mL basic fibroblast growth factor, and 10 µg/mL heparin.  Shear stress on endothelial cell adhesion and spreading was determined using rheological properties of the hydrogel.  We assessed the effect of bioprinting on cell spreading and viability.  Effect of cell density on fiber size was evaluated.  Cells were incubated in growth medium containing 2 µM CFDA-SE at 37°C for 20 min followed by washing the excess stain with growth medium.  CFDA stained cells mixed with hydrogels were printed and cultured.  Cell spreading, and behavior was observed using Nikon Eclipse TE 2000-U microscope.  Digital micrographs were collected from random locations,which were used to determine the fiber sizes using Sigma Scan Pro (SPSS Science, Chicago, IL) image analysis software.  Obtained values were then used to determine the fiber size distribution, average fiber size and standard deviation.  

These results showed that printing of fiber does not have an effect on cell spreading and viability and the size of the fiber can be altered by changing the needle size and cell density.  To understand the use of bioprinting technique, they were incubated for seven days and analyzed for changes in fiber architecture.  These results indicated that the cells were spreading and viable.  Since the primary concern in bioprinting technologies is the effect of environment and shear stress on cells, we also tested the viability of printed cells.  The results showed that HUVECs were viable, attached to the tissue culture plastic surface and spread when observed after one day.  Additional studies need to be done with the use of supporting cells, and growth factors (GFs) to form stable and functional blood vessels.  In summary, we demonstrate that microvessels can be printed using chitosan-gelatin hydrogels while retaining cell viability and spreading characteristics.  Further studies needs to be conducted for testing stability of the newly formed microvessels after co-culturing with supporting cells along with dual GFs delivery system.