Purpose: Poly(gamma-glutamic acid) (gamma-PGA) is an extracellular bacterial polymer with variable molecular weight produced by several members of the genus Bacillus, composed of D and/or L-glutamic acid monomers connected by amide bonds between alpha-amino and gamma-carboxyl groups. Due to its biodegradability and intrinsic absence of toxicity, gamma-PGA meets most of the requirements of polymers for biomedical applications. Gamma-PGA hydrogels are investigated as drug delivery systems for local applications to skin (e.g. wound healing therapy), mucosae (e.g. buccal delivery of anti-infective drugs) as well as biomaterial to make scaffolds for tissue regeneration. The work is aimed to investigate novel gamma-PGA based hydrogels to be processed by 3D bioprinting in order to obtain cellularized scaffold for wound healing.
Methods: Chitosan solutions at 4.5 % and 6 % w/v in acetic acid aqueous solution (1% v/v) (Cs solution, pH 6.0) and 2% w/v Gamma-PGA solution were 3D-bioprinted with Cellink 3D-bioprinter. (Figure 1 a) The polymer solutions were previously characterized for their viscosity at 35°C and 100 sec-1 shear rate that simulate 3D-bioprinter process conditions. Cs solution and 2% Gamma-PGA solution were co-extruded through two different needles and syringes thermostated at 35°C. Uncellularized 3D-bioprinted hydrogel was further crosslinked by dropping on its surface 300 μL of NaOH aqueous solution (1N), hydrogel sample was maintained in basic conditions for 2 min and then it was properly washed with sterile PBS (pH 7.4). Two layers of each Cs solutions (thickness 0.4 mm per layer) were printed in a 10 x10 mm grid, thereafter one layer of 2% Gamma-PGA solution (thickness 0.4 mm per layer) was further printed to obtained a three layer construct hydrogel. Infill tested ranked from 30% and 70%. Pressure was 25 e 40 kPa for 4.5% and 6% w/v Cs solutions respectively, and 5 and 10 kPa for Gamma-PGA 2% solution. Printing rate was maintained at 600 mm/min. Fluid handling capacity and stability of the hydrogels were evaluated during incubation in 10 % DMEM 37°C for 30 days, results were expressed as percentage of water uptake and mass loss. Cellularized hydrogels were prepared loading human adult fibroblasts into the Cs solution (215 kcells/ mL Cs solution). DAPI and Live and Dead tests were performed on cellularized hydrogels to evaluate hydrogel containment ability and cell survival.
Results: Viscosity values of chitosan and Gamma-PGA polymer solutions at 35°C were expressed as torque force, as requested by the bio-printer. All the data in steady state (time zero) and at 100 sec-1 shear rate were below the maximum value allowed by 3D-bioprinter (250 ·10-3 N*m). Torque values of Cs solution (4.5%) at 35°C in steady state and at 100 sec-1 shear rate were 4.00 · E-0.05 N*m and 4.00 E-0.03, respectively; while for Cs solution at 6% w/v torque were 3.382 E-0.04 N*m and 3.276 · E-0.03 N*m. Gamma-PGA solution torque was 7.293 E-0.08 N*m and 2.648 E-0.04 N*m in steady state and dynamic conditions, respectively. Cs solutions at 4.5 and 6% resulted both suitable for 3D-bioprinting with infill 70% (Figure 1b). The obtained hydrogels were stable for 30 days incubation (water uptake 36%, mass loss 14%) and pH of the incubation medium shifted from 8.2 to 7.8 during incubation time. Results of immunostaining with DAPI showed that the cells are embedded in the hydrogel, with about 40% cell survival (Figure 1c).
Morover, results of confocal analysis showed hydrogel porosity in the range 40 -80 microns (Figure 1c).
Conclusion: The preliminary study allows to conclude that chitosan and Gamma-PGA polymer solutions can be 3D-bioprinted as three layer construct hydrogel. The hydrogel is able to embed fibroblasts maintaining its shape and cell loading for up to 30 days. Further characterization is in process to evaluate cell proliferation and perform in vitro characterization of uncellularized and cellularized three layer construct hydrogel.
Rossella Dorati
– Assistant professor, University of PaviaBice Conti
– Professor, University of Pavia, PaviaSilvia Pisani
– PhD student, University of Pavia, PaviaCamilla Mariotti
– Student, University of PaviaIda Genta
– Associate professor, University of PaviaEnrica Chiesa
– Postdoc, University of PaviaTiziana Modena
– Associate professor, University of PaviaFranca Scocozza
– Postdoc, University of PaviaMichele Conti
– Assistant professor, University of PaviaFerdinando Auricchio
– Full professor, University of PaviaBice Conti
– Professor, University of Pavia, Pavia387 Views