Co-3D Printing of Pericyte-supported Pre-vascularized Calcium Phosphate Bone Scaffolds

Objectives: The absence of a vascular network to facilitate the transport of oxygen and nutrients in 3D engineered tissues represents a major hurdle in tissue regeneration. Pre-vascularization of tissue regeneration scaffolds by fabrication of endothelialized microchannels has improved cell survival and function. However, formation of stable, mature and non-leaky vasculature requires support from perivascular cells like pericytes. Differentiation of human Mesenchymal Stem Cells (hMSCs) to pericyte-like lineages and their interactions with Endothelial Cells (ECs) are directed by cues from their microenvironment. The objective of this study is to elucidate the optimal conditions for the formation of stable and mature vasculature. Additionally, we sought to optimize the process of 3D-printing pre-vascularized bone scaffold.
Methods: Gelatin Methacryloyl (GelMA) hydrogels (10%, 15% and 20% (w/v), photo-crosslinked for 30s) were used as substrates for co-cultures of ECs and hMSCs in ratios of 1:1, 4:1 and 1:4 to investigate the effects of EC concentration and matrix stiffness on vasculogenic differentiation of hMSCs. Proliferation was assayed by alamar blue. Cells were stained for F-Actin and DAPI for morphological analysis and immunostained for alpha SMA to probe for pericytic differentiation. A self-curing calcium phosphate bio-ink and sacrificial agarose fibers were co-3D-printed. Subsequently this 3D-printed scaffold was surrounded wtih GelMA hydrogel and photopolymerized. The sacrificial agarose fibers were removed to form microchannels within the construct.
Results: While matrix elasticity did not affect proliferation of 4:1 co-cultures of ECs and hMSCs over a 7-day period, qualitative analysis determined that the softer (10% (w/v) GelMA) substrates were apparently more conducive to vasculogenic differentiation. Also, 4:1 EC: hMSC group had an apparent higher incidence of vasculogenic differentiation than the other groups and hMSCs alone. Microchannels were successfully fabricated within the 3D-printed construct.
Conclusions: Co-culture of ECs with hMSCs in a 4:1 ratio on relatively softer matrices supports vasculogenic differentiation. The fabrication process for 3D-printed constructs was optimized. Our future work involves the seeding of co-cultured cells within the microchannels of the 3D-printed construct.