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"Introduction
Annually, millions of people die because of liver failure, while the waiting duration for a donor liver is around 12 months.[1] Herein, we target hybrid 3D-printed scaffolds to serve liver tissue engineering(LTE) applications.
As starting materials gelatin in combination with a polysaccharide was used to develop printable hydrogels. As polysaccharides dextran (Dex) and chondroitin sulphate (CS) were selected as mimics for the liver extracellular matrix (ECM) to explore their effect on the cell response. Methacrylated gelatin (GelMA) served as benchmark. The hydrogel materials were characterized on 2D-as well as on 3D-level.
HepG2 cells were used to assess the in vitro biocompatibility of the developed hydrogels.
Materials and methods
Thiolated gelatin (GelSH)[2] was crosslinked with the norbornene-functionalized polysaccharides DexNB and CSNB. Gelatin was methacrylated using methacrylic anhydride (GelMA) as reference material.
The different materials were characterized on both 2D- and 3D-level to assess the physico-chemical properties and the biocompatility. 3D-hydrogel scaffolds of the materials were developed by indirect 3D-printing[3].
Results and discussion
On a 2D-level, DexNB-GelSH and CSNB-GelSH were superior over GelMA as their crosslinking kinetics were significantly faster and they mimicked natural liver tissue (NLT) to a greater extent with respect to swelling and mechanical properties. The swelling ratio of GelMA, DexNB-GelSH and CSNB-GelSH were respectively 9.1±0.5 and 9.6±0.5 and 8.7±0.2 which is in line with the swelling of NLT (i.e.10)[3].
Atomic Force Microscopy (AFM) measurements revealed superior microscale mechanical properties of the DexNB-GelSH hydrogel sheets compared to the other materials. DexNB-GelSH exhibited a stiffness of (196±24)kPa, CSNB-GelSH of (106±2)kPa and GelMA of (291±11)kPa. Healthy NLT exhibits an average surface stiffness of (183±48)kPa. The higher the stiffness, the more the material mimics the ECM of unhealthy cirrhotic liver ((411± 63)kPa)[4].
On a 3D-level, DexNB-GelSH scaffolds exhibited a compressive modulus of (4.8±1.6)kPa which is in excellent agreement with that of NLT (i.e. 1–5kPa)[5] as compared to GelMA which resulted in a modulus of (8.5±1.9)kPa and CSNB-GelSH (12.6±1.9)kPa.
So far, the biocompatibility of CSNB-GelSH and GelMA were compared. However, the in vitro biocompatibility of both materials was comparable based on the MTS assay. The live-dead staining showed that the cells grew more into clusters on the DexNB-GelSH scaffolds compared to the more spread morphology which the cells exhibited on the GelMA material.
Conclusions and future perspectives
DexNB-GelSH and CSNB-GelSH scaffolds are promising hybrid materials to support LTE as they exhibit similar physico-chemical properties compared to NLT (cfr. microscale stiffness, compressive modulus, swelling ratio and chemical compostion), while cell viability and proliferation of the hepatocytes were preserved.
In future research, different cell types such as primary hepatocytes and organoids will be included in the biological evaluation. Furthermore decellularized liver ECM will be incorporated into the hydrogel material in order to improve the cell interactivity and proliferation.
1)Emek, E. et al. Transplant. Proc. 51 (7), 2413-2415(2019)
2)Van Vlierberghe, S. et al. Eur. Polym. J. 47 (5), 1039-1047(2011)
3)Mattei, G. et al. Acta Biomater. 10, 875–882(2014)
4)Zhao, G. et al. J. Surg. Oncol. 102, 482-489(2010)
5)Mattei, G. ACTA Biomater. 10(2), 875-882(2014)"
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