Biology subjects

Putnam, A. J.

Publications and source records attributed to Putnam, A. J..

5 recordsLinked to original sources

bioRxiv2026

Ultrasound-reconfigurable scaffolds enable dynamic control of biochemical and biophysical cues for vascular network formation

Vascularization, which is critical for most engineered tissue constructs, is dependent on interactions between endothelial cells and spatiotemporally presented biochemical and biophysical cues. Yet, most hydrogels define these cues at the time of fabrication, thus precluding adjustments to actively drive vascular formation. We developed acoustically responsive scaffolds (ARSs) that use focused ultrasound to trigger growth factor release and localized matrix remodeling within fibrin hydrogels. ARSs were formed by incorporating phase-shift emulsions containing basic fibroblast growth factor (bFGF) along with perfluorohexane (C6) or perfluorooctane (C8). Upon ultrasound exposure, stable bubbles were generated in C6-ARSs that locally compacted the matrix and increased macroscale stiffness. Comparatively, in C8-ARSs, ultrasound generated macropores without impacting viscoelastic properties. Ultrasound increased bFGF release from both ARS types, which enhanced in vitro and in vivo vasculogenic assembly in ARSs with co-encapsulated endothelial cells and fibroblasts. Our data also show that ultrasound-driven matrix remodeling without bFGF release increased endothelial sprouting. In C6-ARSs, elevated levels of F-actin were observed in both cell types adjacent to bubbles as well as increased YAP intensity and nuclear asymmetry. Together, these results establish ARSs as reconfigurable hydrogels that couple on-demand release of biochemical cues with programmable matrix restructuring to direct three-dimensional microvascular assembly.

bioengineering↗

bioRxiv2026

A robust and user-agnostic step-emulsion platform for scalable microgel fabrication

Over the past decade, the integration of microgel-based granular hydrogels in biomedical technologies has experienced substantial growth due to the numerous benefits microgels offer. However, the inability to easily adopt uniform microgel fabrication workflows at scale constitutes a major bottleneck, or in some cases, a barrier-to-entry that stunts further growth of the field. The gold-standard technique for emulsion-based microgel production is through microfluidic droplet-generating devices that produce liquid gel precursor droplets that gel post-production. However, traditional microfluidic workflows often require multiple independent flows and controlled pressure sources, along with a steep learning curve in using microfluidics to achieve uniform droplet sizes reproducibly and repeatedly. This difficulty in adopting microgel fabrication is further compounded by low throughput and the extensive flow rate calibration required when switching to new formulations (e.g., material type, droplet size). In this work, we present a step-emulsion system that bridges the gap by providing a robust and simple setup. We experimentally characterize and evaluate how flow and outlet channel dimension contribute to the generation of uniform droplet populations at specific sizes. With our large dataset consisting of various outlet channel dimensions, we evaluated outlet channel geometrical impacts (height, width, cross-sectional area, aspect-ratio, etc.) on gel precursor droplet size and generation throughput. We demonstrate robust, highly compatible, and repeatably uniform droplet generation from various gel precursor polymer backbones, users with varying microfluidics experience, and a wide viscosity range, including alginate solutions with 650 times the viscosity of water. Furthermore, we confirmed consistent gel precursor droplet generation outcomes driven by a constant flow source (syringe pump) and by direct manual injection as a simple and highly adoptable option for the generation of gel precursor droplets. This platform is ideal for researchers seeking rapid and easy microgel fabrication, regardless of microfluidics experience.

bioengineering↗

bioRxiv2025

Amoeboid-Mesenchymal Transition and the Proteolytic Control of Cancer Invasion Plasticity

Invasion plasticity allows malignant cells to toggle between collective, mesenchymal and amoeboid phenotypes while traversing extracellular matrix (ECM) barriers. Current dogma holds that collective and mesenchymal invasion programs trigger the mobilization of proteinases that digest structural barriers dominated by type I collagen, while amoeboid activity allows cancer cells to marshal mechanical forces to traverse tissues independently of ECM proteolysis. Here, we use cancer spheroid-3-dimensional matrix models, single-cell RNA sequencing, and human tissue explants to identify the mechanisms controlling mesenchymal versus amoeboid invasion. Unexpectedly, collective/mesenchymal- and amoeboid-type invasion programs - though distinct - are each characterized by active tunneling through ECM barriers, with expression of matrix-degradative metalloproteinases. CRISPR/Cas9-mediated targeting of a single membrane-anchored collagenase, MMP14/MT1-MMP, ablates tissue-invasive activity while co-regulating cancer cell transcriptional programs. Though changes in matrix architecture, nuclear rigidity, and metabolic stress as well as the presence of cancer-associated fibroblasts are proposed to support amoeboid activity, none of these changes restore invasive activity of MMP14-targeted cancer cells. While a requirement for MMP14 is bypassed in low-density collagen hydrogels, invasion by the proteinase-deleted cells is associated with nuclear envelope and DNA damage, highlighting a proteolytic requirement for maintaining nuclear integrity. Nevertheless, when cancer cells confront explants of live human breast tissue, MMP14 is again required to support invasive activity. Corroborating these results, spatial transcriptomic and immunohistological analyses of invasive human breast cancers identified clear expression of MMP14 in invasive cells that were further associated with degraded collagen, underlining the pathophysiologic importance of this proteinase in directing invasive activity in vivo.

cancer biology↗

bioRxiv2024

Clickable PEG-norbornene microgels support suspension bioprinting and microvascular assembly

The development of perfusable and multiscale vascular networks remains one of the largest challenges in tissue engineering. As such, there is a need for the creation of customizable and facile methods to produce robustly vascularized constructs. In this study, secondarily crosslinkable (clickable) poly(ethylene glycol)-norbornene (PEGNB) microbeads were produced and evaluated for their ability to sequentially support suspension bioprinting and microvascular self-assembly towards the aim of engineering hierarchical vasculature. The clickable PEGNB microbead slurry exhibited mechanical behavior suitable for suspension bioprinting of sacrificial bioinks, could be UV crosslinked into a granular construct post-print, and withstood evacuation of the bioink and subsequent perfusion of the patterned void space. Endothelial and stromal cells co-embedded within jammed RGD-modified PEGNB microbead slurries assembled into capillary-scale vasculature after secondary crosslinking of the beads into granular constructs, with endothelial tubules forming within the interstitial space between microbeads and supported by the perivascular association of the stromal cells. Microvascular self-assembly was not impacted by printing sacrificial bioinks into the cell-laden microbead support bath before UV crosslinking. Collectively, these results demonstrate that clickable PEGNB microbeads are a versatile substrate for both suspension printing and microvascular culture and may be the foundation for a promising methodology to engineer hierarchical vasculature.

bioengineering↗

bioRxiv2018

Non-adhesive alginate hydrogels support growth of pluripotent stem cell-derived intestinal organoids

Human intestinal organoids (HIOs) represent a powerful system to study human development and are promising candidates for clinical translation as drug-screening tools or engineered tissue. Experimental control and clinical use of HIOs is limited by growth in expensive and poorly defined tumor-cell-derived extracellular matrices, prompting investigation of synthetic ECM-mimetics for HIO culture. Since HIOs possess an inner epithelium and outer mesenchyme, we hypothesized that adhesive cues provided by the matrix may be dispensable for HIO culture. Here, we demonstrate that alginate, a minimally supportive hydrogel with no inherent cell adhesion properties, supports HIO growth in vitro and leads to HIO epithelial differentiation that is virtually indistinguishable from Matrigel-grown HIOs. Additionally, alginate-grown HIOs mature to a similar degree as Matrigel-grown HIOs when transplanted in vivo, both resembling human fetal intestine. This work demonstrates that purely mechanical support from a simple-to-use and inexpensive hydrogel is sufficient to promote HIO survival and development.

developmental biology↗