Recent advancements in 3D bioprinting have led to the fabrication of more complex, more precise, and larger printed tissue constructs. As the field continues to advance, it is critical to develop quantitative benchmarks to compare different bio-inks for key cell-biomaterial interactions, including (1) cell sedimentation within the ink cartridge, (2) cell viability during extrusion, and (3) cell viability after ink curing. Here we develop three simple protocols for quantitative analysis of bio-ink performance. These methods are used to benchmark the performance of two commonly used bio-inks, poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), against three formulations of a novel bio-ink, Recombinant-protein Alginate Platform for Injectable Dual-crosslinked ink (RAPID ink). RAPID inks undergo peptide-self-assembly to form weak, shear-thinning gels in the ink cartridge and undergo electrostatic crosslinking with divalent cations during curing. In the one-hour cell sedimentation assay, GelMA, the RAPID inks, and PEGDA with xanthan gum prevented appreciable cell sedimentation, while PEGDA alone or PEGDA with alginate experienced significant cell settling. To quantify cell viability during printing, 3T3 fibroblasts were printed at a constant flowrate of 75 μl/min and immediately tested for cell membrane integrity. Less than 10% of cells were damaged using the PEGDA and GelMA bio-inks, while less than 4% of cells were damaged using the RAPID inks. Finally, to evaluate cell viability after curing, cells were exposed to ink-specific curing conditions for five minutes and tested for membrane integrity. After exposure to light with photo-initiator at ambient conditions, over 50% of cells near the edges of printed PEGDA and GelMA droplets were damaged. In contrast, fewer than 20% of cells found near the edges of RAPID inks were damaged after a 5-minute exposure to curing in a 10 mM CaCl2 solution. As new bio-inks continue to be developed, these protocols offer a convenient means to quantitatively benchmark their performance against existing inks.

Current bioinks for cell-based 3D bioprinting are not suitable for technology scale-up due to the challenges of cell sedimentation, cell membrane damage, and cell dehydration. A novel bioink hydrogel is presented with dual-stage crosslinking specifically designed to overcome these three major hurdles. This bioink enables the direct patterning of highly viable, multicell type constructs with long-term spatial fidelity.

Transplantation of patient-derived Schwann cells is a promising regenerative medicine therapy for spinal cord injuries; however, therapeutic efficacy is compromised by inefficient cell delivery. We present a materials-based strategy that addresses three common causes of transplanted cell death: (i) membrane damage during injection, (ii) cell leakage from the injection site, and (iii) apoptosis due to loss of endogenous matrix. Using protein engineering and peptide-based assembly, we designed injectable hydrogels with modular cell-adhesive and mechanical properties. In a cervical contusion model, our hydrogel matrix resulted in a greater than 700% improvement in successful Schwann cell transplantation. The combination therapy of cells and gel significantly improved the spatial distribution of transplanted cells within the endogenous tissue. A reduction in cystic cavitation and neuronal loss were also observed with substantial increases in forelimb strength and coordination. Using an injectable hydrogel matrix, therefore, can markedly improve the outcomes of cellular transplantation therapies.

The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGF␣, neuregulin 1␤ (NRG), and heparinbinding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substratespecific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGF␣ and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKC enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.Metalloprotease-mediated ectodomain cleavage of transmembrane proteins such as EGF ligand precursors is involved in the regulation of many physiological signaling pathways, and its dysregulation can cause kidney disease, heart disease, and cancer (1, 2). For example, in the kidney, many of the deleterious effects of chronic exposure to the G-protein-coupled receptor (GPCR) 2 agonist angiotensin II are mediated by metalloproteinase (ADAM17)-dependent release of the EGF ligand TGF␣ and subsequent epidermal growth factor receptor activation. Angiotensin II is the main effector of the renin-angiotensin system and has important roles in the regulation of blood pressure and aldosterone secretion (3).Although several ADAM gene disruptions are lethal in the mouse, metalloprotease inhibitors have shown therapeutic potential; however, not unexpectedly, the essential role of ADAMs in normal physiology as well as the broad spectrum nature of currently available inhibitors are probably the cause for a number of their side effects. In addition, concerns have been raised that some metalloproteases act as tumor suppressors (4,5). A more precise inhibiti...