Abstract
Purpose :
3D bio-printing makes precisely controlled deposition of cell-laden hydrogels possible, producing an artificial biomimetic corneal substitute. Within this study, indirect functionalization of alginate fibers via the addition of gelatin or 2-Hydroxyethyl cellulose (HEC) in the crosslinking bath was examined, geometric properties and extrudability were characterized. This was with the aim of improving print resolution, fidelity and growth characteristics of encapsulated cells.
Methods :
Human limbal stromal fibroblasts (hLSFs) grown in standard serum containing medium were detached, mixed with 1.2% alginate and extruded through a 23G needle at 1mL/min into 0.1M calcium chloride (CaCl2) crosslinking bath with or without gelatin or HEC (1, 3, 5 and 10%) to produce alginate fibers, followed by a 5 minute washing step with HBSS before capturing images for subsequent analysis. Methodical characterization of diameter, length and volume was performed. Gelatin integration was examined using Sirius Red assay. The viability of encapsulated hLSFs within the fibers was evaluated using cell titer glo assay over 7 days and compared against non-encapsulated hLSFs.
Results :
An inversely proportional relationship between the length and diameter of the produced fibers was observed. Fiber length decreased whilst fiber diameter increased in response to both gelatin and HEC in a concentration-dependent manner (p = < 0.001; n = 3). Sirius Red data demonstrated increased collagen accumulation when exposed to 3%-10% gelatin compared to the control, (p = < 0.0001; n = 3). Encapsulated hLSF remained viable over 7 days in culture.
Conclusions :
The nature of the crosslinking bath, that the bio-ink is extruded into, can have a considerable effect on the properties of the fibers. Through incorporating gelatin and HEC into the crosslinking bath, gelation kinetics were modified. This effect was dependent on viscosity of the crosslinking bath and could be mitigated, in part, by increasing the extrusion speed.
Interestingly, crosslinking baths containing gelatin increased collagen accumulation in the fibers, yet encapsulated cells exhibited minimal cell-matrix interaction at concentrations tested. For future corneal bio-printing applications, the properties of the crosslinking solution should be considered critical to control the printed fiber diameter, which in turn influences the final resolution of the printed structure.
This is a 2020 ARVO Annual Meeting abstract.