Hydrogel bioprinted microchannel networks for vascularization of tissue engineering constructs

Abstract

Vascularization remains a critical challenge in tissue engineering. The development of vascular networks within densely populated and metabolically functional tissues facilitate transport of nutrients and removal of waste products, thus preserving cellular viability over a long period of time. Despite tremendous progress in fabricating complex tissue constructs in the past few years, approaches for controlled vascularization within hydrogel based engineered tissue constructs have remained limited. Here, we report a three dimensional (3D) micromolding technique utilizing bioprinted agarose template fibers to fabricate microchannel networks with various architectural features within photocrosslinkable hydrogel constructs. Using the proposed approach, we were able to successfully embed functional and perfusable microchannels inside methacrylated gelatin (GelMA), star poly(ethylene glycol-co-lactide) acrylate (SPELA), poly(ethylene glycol) dimethacrylate (PEGDMA) and poly(ethylene glycol) diacrylate (PEGDA) hydrogels at different concentrations. In particular, GelMA hydrogels were used as a model to demonstrate the functionality of the fabricated vascular networks in improving mass transport, cellular viability and differentiation within the cell-laden tissue constructs. In addition, successful formation of endothelial monolayers within the fabricated channels was confirmed. Overall, our proposed strategy represents an effective technique for vascularization of hydrogel constructs with useful applications in tissue engineering and organs on a chip.

Figure 1

schematic representation of bioprinting of agarose template fibers and subsequent formation of microchannels via template micromolding. a) a bioprinter equipped with a piston fitted inside a glass capillary aspirates the agarose (inset). after gelation in 4°c, agarose fibers are bioprinted at predefined locations. b) a hydrogel precursoris casted over the bioprinted mold and photo cross linked. c) the template is removed from the surrounding photo cross linked gel. d) fully perfusable microchannels are formed.

Figure 2

hydrogel properties associated with microchannel formation. a) reproducibility of microchannel formation using 2 to 8% agarose templates in 5 to 20% gelma, pegda, spela and pegdma hydrogels. filled-squares represent successful microchannel formation, while crossed-squares represent failed microchannel formation. 5% pegdma and spela did not cross link. b) stress and strain response of hydrogels under compressive loading. c) 20% pegda hydrogels showed significantly higher modulus than 20% pegdma (*p<0.05), 20% spela and 20% gelma hydrogels (****p<.0001). d) 5% pegda hydrogels had higher mass swelling ratio than 5% gelma hydrogels (***p<0.001), while 10% gelma hydrogels had a higher swelling ratio than pegdma hydrogels (*p<0.05). e) representative image of microchannels with approximately 1000 μm,500 μm and 150 μm (left to right) in diameter are shown (500 μm scale bar).

Figure 3

bioprinted agarose template fibers and respective microchannels. a) linear parallel agarose template fibers (scale bar 1 mm) and (a–i) cross-section (indicated by red dotted-line) perspective of a fluorescent microbead-laden gelma hydrogel showing the circular shape of the lumen (scale bar 250 μm). circular morphology of microchannels from additional gels is shown in supplementary figure s3. b) planar bifurcating agarose template fibers (scale bar 1 mm) and (b–i) cross-section perspective (red dotted-line) of microbead-laden gelma hydrogel microchannels showing the interconnectivity of the bifurcating network (scale bar 250 μm). to p view perspective of planar bifurcating network is shown in supplementary figure s4. c) three dimensional lattice architecture of perpendicular bioprinted fibers (scale bar 1 mm) and (c–i) cross-section perspective (red dotted-line) of the network molded from lattice template. white lines indicate the upper and lower boundaries of the microchannel (scale bar 500 μm). d-diii) a sequence of images of a gelma hydrogel chip with integrated microchannels perfused from one inlet (lower microchannel) illustrates the fluid flow through the entire construct (scale bar 500 μm).

Figure 4

photographs of the bioprinted templates (green) enclosed in gelma hydrogels and the respective microchannels perfused with a fluorescent microbead suspension (pink). a) planar bifurcating bioprinted templates in a gelma hydrogel construct and (a-i) respective network after perfusion. b) 3d branching agarose templates embedded in agelma hydrogel construct and (b-i) resulting 3d branching network. c) 3d lattice template embedded in a gelma hydrogel and (c-i) after perfusion. (scale bars 3 mm, microchannels have 500 μmin diameter)

Figure 5

viability and differentiation of mc3t3 cells encapsulated in 10% gelma hydrogels comparing constructs with fabricated microchannels versus blocks without microchannels. live and dead images of gelma hydrogel blocks (a and b) and hydrogels with fabricated microchannels (c and d) at days 1 and 7. e) hydrogel blocks had significantly lower viability at day 1 (*p<0.05) and day 7 (***p<0.001). f) alp specific activity assay showed significantly higher alp activity levels in cell-laden constructs with microchannels versus cell-laden hydrogel blocks on day 14 (****p<0.0001).(scale bar 700 μm)

Figure 6

representative confocal and fluorescent microscopy images of immunostained huvecs forming a monolayer inside microchannels of different diameters after 7 days. a) proliferation of huvecs in 250 μm,500 μm compared to 1000 μm channels (***p<0.001, **p<0.01 and *p<0.05). b) 1000 μm,(c) 500 μm and (d) 250 μm microchannels lined with endothelial cells (scale bars 250 μm). e) photograph of vascularized gelma hydrogel construct with mature endothelial monolayer visible by naked eye covering the entire length of the channels. f) confocal image of gfp/dapi/cd31 markers from a huvec monolayer inside a 500 μm channel (scale bar 250 μm). g) higher magnification fluorescence image of dapi- and cd31- stained gfp-expressing huvecs illustrating the cell-cell interactions (arrowheads) along the endothelial monolayer (scale bar 50 μm). h) longitudinal view of z-stacked confocal images of a huvec-lined microchannel. the inset shows across-section view of the channel (scale bars 250 μm). perpendicular views of z-stacked (i) dapi, (j) cd31 and (k) gfp markers are also shown to illustrate the complete lining of the microchannel lumen (scale bars 250 μm). three dimensional animations of the respective still images are shown as supplementary movies 5a–5e.