Our newly developed self-feeding hydrogels with enzyme-empowered degradation capacity have demonstrated high biological performance in-vitro and in-vivo as a novel self-maintained and biocompatible 3D scaffold1. Photo-crosslinkable platelet lysates (PL)-based hydrogels have exhibited to support distinct human-derived cell cultures owing to their high content of bioactive molecules, such as cytokines and growth factors2. To take advantage of all features of both PL and self-feeding hydrogels, here we combined UV responsive laminaran-methacrylate (MeLam) and PL-methacrylate (PLMA) derivatives plus glucoamylase (GA) to fabricate a multicomponent hybrid hydrogel (GLMPL). This hydrogel emerged as an unique scaffold due to the combination of sustained delivery of glucose produced via enzymatic degradation of laminaran and granting cell adhesin by presence of PL. Besides, this biomaterial was also applied to fabricate high-throughput freestanding microgels with controlled geometrically shapes. Impressively, such multicomponent hybrid hydrogel was successfully implemented as a glucose supplier bioink to fabricate complex and well-defined cell-laden structures using a support matrix.
MeLam and PLMA were synthesized following the previous reports2,3. In order to obtain a gradual production of glucose over time, GA enzyme was incorporated into MeLam/PLMA mixtures before UV exposure. We applied superhydrophobic surfaces (SH) patterned with wettable shaped domains (SL)4, where suspensions of cells and GLMPL hydrogel precursor could be dispersed and microgels with different geometries were produced after UV irradiation. To demonstrate the outstanding bifunctionality of such bioink, a mixture of GLMPL hydrogel precursor and cells suspension were deposited into agarose support matrix by extrusion 3D-printer. Thereafter the printed structures were exposed to UV to form gels. In vitro studies were performed on these biomaterials by encapsulating Human adipose-derived stem cells (hASCs) cultured in glucose free Dulbecco's Modified Eagle Medium. As such, any difference in cells response could then be attributed directly to the presence of enzyme and consequently glucose accessibility for the encapsulated cells.
CellTiter-Glo assay has shown hASCs metabolic activity significantly increased in GLMPL hydrogels over 21 days. Moreover, pronounced cell proliferation was confirmed through DNA quantification. Live-Dead assay confirmed encapsulated hASCs stretched inside the GLMPL hydrogel. DAPI/phalloidin staining have approved that cells elongated in GLMPL hydrogels and formed interconnected networks with neighbouring cells. Live-dead assay showed that up to 7 days of culture, most of the hASCs remained viable and elongated inside the microgels and 3D printed structures. It is noteworthy to mention that freestanding microgels and bioprinted scaffolds showed well-preserved architecture during the culture.
In conclusion, these results, combined that most current bioscaffolds suffer from lack of nutrient diffusion and adhere motifs, clearly suggest the potential of this multifunctional hybrid hydrogel in future developments of 3D structures in a wide range of biotechnological applications as an autonomous cell supporting system.
Authors acknowledge the project CICECO-Aveiro Institute of Materials UIDB/50011/2020 & UIDP/50011/2020, SFRH/BD/143883/2019 and CEECIND/02713/2017.
1. Zargarzadeh, M., et al., Materials Horizons (2022).
2. Santos, S.C., Custódio, C.A. & Mano, J.F, Advanced Healthcare Materials 7, 1800849 (2018).
3. Custódio, C.A., Reis, R.L. & Mano, J.F. , Biomacromolecules 17, 1602-1609 (2016).
4. Neto, A.I., et al., Advanced Materials 28, 7613-7619 (2016).