3D printing of thermo-responsive hydrogels

by Silvia Faré, Associate Professor, Politecnico di Milano – Dept Chemistry, Materials and Chemical Engineering

Smart hydrogels reversibly change their properties when exposed to an external driven force as pH or temperature variation (1). Methylcellulose (MC) is a polysaccharide derived from cellulose and when dissolved in aqueous solvents it forms reverse thermo-responsive smart hydrogels that undergo a sol-gel transition when heated (2).

At the same time, another smart material is represented by gelatin that needs to be crosslinked for biomedical applications. Crosslinked gelatin bulk scaffolds, 3D printed structures and microspheres can be obtained by tuning its crosslinking kinetic, innovatively without the need of post-curing or external treatments to stabilize the crosslinked printed hydrogel structure (3).

Possible applications in regenerative medicine of MC-based hydrogels as well as crosslinked gelatin hydrogels are here described.

Materials and Methods
MC-based hydrogels. METHOCEL powder 8% w/v was dissolved at 55 °C in different distilled water saline solutions: phosphate buffered saline at two different concentrations (MC_PBS10 and MC_PBS20), sodium sulfate 0.05 M (MC_Na005) and 0.1 M (MC_Na01) and distilled water as control (MC_water). After complete powder dissolution, solutions were sealed in petri dishes and stored at 4 °C for 24 h to allow MC complete hydration (2).

Crosslinked gelatin hydrogels. Gelatin hydrogels (GEL) were crosslinked by a Michael-type addition by mixing gelatin type A from porcine skin (15% w/v) and methylenebisacrylamide, MBA, used as crosslinker (23.3 mg), to initiate the crosslinking reaction. Different hydrogels were prepared by varying gelatin concentration (i.e., 15 or 25% w/v) and the crosslinking reaction stoichiometry (i.e., MBA:gelatin amines = 1:1 or 0.5:1).

MC and gelatin hydrogels were investigated by investigating their chemico-physical, mechanical and in vitro biological properties. 3D printing was also investigated for both the smart hydrogels.

Results and Discussion
MC-based hydrogels. Rheological and UV-spectroscopy tests showed that the addition of salts to MC hydrogels allowed lowering the LCST of the MC hydrogel; moreover, hydrogels produced in 0.1 M Na2SO4 or PBS 20 g/L were proved to be particularly promising for cell sheet engineering application, showing a LCST below 37 °C. Extrusion-based 3D printing was shown to be an effective strategy for cell sheet engineering, having a desired shape using MC-based hydrogels as ink, with the ultimate goal of the regeneration of complex tissues.

Crosslinked gelatin hydrogels. The physico-mechanical properties of chemically crosslinked GELs were successfully tuned by varying gelatin concentration and reaction stoichiometry and allowed identifying the most suitable formulation (15GEL05) as soft tissue substitute. Moreover, the cytocompatibility of GELs was proved and the ability of GELs in supporting cells attachment and proliferation make the proposed hydrogel a valid material as soft tissue substitute. 15GEL05 was successfully printed by tuning the crosslinking kinetic the printing process optimization. The obtained 3D porous scaffolds proved to be suitable as supports for soft tissue regeneration.

Conclusion
Smart hydrogels showed appropriate properties for applications in regenerative medicine by tuning their properties.

References: 1. L. Klouda. Eur. J. Pharm. Biopharm. 97 (2015) 338–349. 2. L. Altomare, et al. J. Mater. Sci. Mater. Med. 27 (2016). 3. Tanzi MC et al., WO2012164032 A1 Patent.

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