Macromolecular modulation of a 3D hydrogel construct differentially regulates human stem cell tissue-to-tissue interface

The simultaneous generation of multiple tissues and their functional assembly into complex tissues remains a critical challenge for regenerative medicine. The tissue-to-tissue interface connecting two adjacent tissues is vital in effective tissue function. The presented worked hypothesize that differential functional property can be engineered by modulating the macromolecular composition of a 3D hydrogel construct and distinctively endow stem cell fate. Hence, it was possible to successfully generate macromolecular constructs by using the extracellular matrix (ECM)-based materials; type I collagen (Col I) and hyaluronic acid (HA); and natural-derived biomaterials as methacrylated gellan-gum (GGMA). The 3D hydrogel constructs consisted of two dissimilar layers: 1) Col I: HA hydrogel and 2) GGMA hydrogel. The tissue-to-tissue interface was created by seeding human mesenchymal stem cells (MSCs) between the two layers. Differential functional rheological and mechanical properties characterized the acellular 3D gradient hydrogel constructs. The cell-based 3D hydrogel constructs were assessed for MSCs viability by live/dead staining. Assessing apoptosis by flow cytometry, data showed the feasibility of the 3D hydrogel constructs in maintaining cell viability with no apoptosis induction onto MSCs. A homogeneous distribution was achieved in a successful cellular tissue-to-tissue interface. Human MSCs low proliferative rate and low ECM deposition were seen for all constructs; however, lower proliferative rate within the ECM microenvironment highlights controlled self-renewal of MSCs. The 3D hydrogel constructs maintained the human MSCs phenotype, yet the macromolecular modulation allowed tuning the human MSCs morphology from round to spindle-shaped phenotype. The intrinsic properties of the 3D cell-based hydrogel construct induced differential inflammatory and angiogenic paracrine secretory profiles owing to the dissimilar engineered biophysical milieu. Human MSCs sense the nearby macromolecular environment adjusting the cell-ECM interactions, which influence cell behaviour and fate. Beyond multi-tissue regeneration, the engineered cellular 3D hydrogel constructs may simultaneously address immune regeneration.This material is based on works supported by the European Union funding under the 7th Framework Programme under Grant Agreement Number 317304 and supported in part by a research grant from Science Foundation Ireland (SFI) and co-funded under the European Regional De-velopment Fund under Grant Number 13/RC/2073_P2. D.R. Pereira ac-knowledges the Foundation for Science and Technology (FCT) , Portugal, for an individual grant (SFRH/BD/81356/2011) . J. Silva-Correia and J.M. Oliveira also acknowledges the FCT for the funds provided under the prestigious program Investigator FCT 2015 (IF/00115/2015 and IF/01285/2015, respectively).The authors acknowledge the scientific and technical assistance of the Electron Microscopy unit within the Centre for Microscopy & Imaging, the flow cytometry core facility and histology core facility at the National University of Ireland (NUI)Galway. All facilities are funded by NUI Galway and the Irish Government's Programme for Research in Third Level Institutions (PRTLI), Cycles 4 and 5, National Development Plan 2007-2013. The authors would like to also acknowledge the Centre for Microscopy & Imaging funded by the National University of Ireland Galway and MSSI in the University of Limerick for the rheometer facilities, both funded by the Programme for Research in Third-Level Institutions (PRTLI), Cycles 4 and 5, National Development Plan 2007-2013