Cole A. DeForest

Dynamically tunable cell culture platforms for tissue engineering and mechanobiology

Human tissues are sophisticated ensembles of many distinct cell types embedded in the complex, but well-defined, structures of the extracellular matrix (ECM). Dynamic biochemical, physicochemical, and mechano-structural changes in the ECM define and regulate tissue-specific cell behaviors. To recapitulate this complex environment in vitro, dynamic polymer-based biomaterials have emerged as powerful tools to probe and direct active changes in cell function. The rapid evolution of polymerization chemistries, structural modulation, and processing technologies, as well as the incorporation of stimuli-responsiveness, now permit synthetic microenvironments to capture much of the dynamic complexity of native tissue. These platforms are comprised not only of natural polymers chemically and molecularly similar to ECM, but those fully synthetic in origin. Here, we review recent in vitro efforts to mimic the dynamic microenvironment comprising native tissue ECM from the viewpoint of material design. We also discuss how these dynamic polymer-based biomaterials are being used in fundamental cell mechanobiology studies, as well as towards efforts in tissue engineering and regenerative medicine.

Multicellular Vascularized Engineered Tissues through User-Programmable Biomaterial Photodegradation

A photodegradable material-based approach to generate endothelialized 3D vascular networks within cell-laden hydrogel biomaterials is introduced. Exploiting multiphoton lithography, microchannel networks spanning nearly all size scales of native human vasculature are readily generated with unprecedented user-defined 4D control. Intraluminal channel architectures of synthetic vessels are fully customizable, providing new opportunities for next-generation microfluidics and directed cell function.

Photomediated oxime ligation as a bioorthogonal tool for spatiotemporally-controlled hydrogel formation and modification

Click chemistry has proved a valuable tool in biocompatible hydrogel formation for 3D cell culture, owing to its bioorthogonal nature and high efficiency under physiological conditions. While traditional click reactions can be readily employed to create uniform functional materials about living cells, their spontaneity prohibits spatiotemporal control of material properties, thereby limiting their utility in recapitulating the dynamic heterogeneity characteristic of the in vivo microenvironment. Photopolymerization-based techniques gain this desired level of 4D programmability, but often at the expense of introducing propagating free radicals that are prone to non-specific reactions with biological systems. Here we present a strategy for bioorthogonal hydrogel formation and modification that does not rely on propagating free radicals, proceeding through oxime ligation moderated by a photocaged alkoxyamine. Upon mild near UV light exposure, the photocage is cleaved, liberating the alkoxyamine and permitting localized condensation with an aldehyde. Multi-arm crosslinkers, functionalized with either benzaldehydes or photocaged alkoxyamines, formed oxime-based hydrogels within minutes of light exposure in the presence of live cells. Polymerization rates and final mechanical properties of these gels could be systematically tuned by varying crosslinker concentrations, light intensity, aniline catalyst equivalents, and pH. Moreover, hydrogel geometry and final mechanical properties were controlled by the location and extent of UV exposure, respectively. Photomediated oxime ligation was then translated to the biochemical modification of hydrogels, where full-length proteins containing photocaged alkoxyamines were immobilized in user-defined regions exposed to UV light. The programmability afforded by photomediated oxime ligation can recapitulate dynamically anisotropic mechanical and biochemical aspects of the native extracellular matrix. Consequently, photopolymerized oxime-based hydrogels are expected to enable an enhanced understanding of cell-matrix interactions by serving as improved 4D cell culture platforms.

A Combinational Effect of “Bulk” and “Surface” Shape-Memory Transitions on the Regulation of Cell Alignment

A novel shape-memory cell culture platform has been designed that is capable of simultaneously tuning surface topography and dimensionality to manipulate cell alignment. By crosslinking poly(ε-caprolactone) (PCL) macromonomers of precisely designed nanoarchitectures, a shape-memory PCL with switching temperature near body temperature is successfully prepared. The temporary strain-fixed PCLs are prepared by processing through heating, stretching, and cooling about the switching temperature. Temporary nanowrinkles are also formed spontaneously during the strain-fixing process with magnitudes that are dependent on the applied strain. The surface features completely transform from wrinkled to smooth upon shape-memory activation over a narrow temperature range. Shape-memory activation also triggers dimensional deformation in an initial fixed strain-dependent manner. A dynamic cell-orienting study demonstrates that surface topographical changes play a dominant role in cell alignment for samples with lower fixed strain, while dimensional changes play a dominant role in cell alignment for samples with higher fixed strain. The proposed shape-memory cell culture platform will become a powerful tool to investigate the effects of spatiotemporally presented mechanostructural stimuli on cell fate.

Light-Activated Proteomic Labeling via Photocaged Bioorthogonal Non-Canonical Amino Acids

This work introduces light-activated bioorthogonal noncanonical amino acid tagging (laBONCAT) as a method to selectively label, isolate, and identify proteins newly synthesized at user-defined regions in tissue culture. By photocaging l-azidohomoalanine (Aha), metabolic incorporation into proteins is prevented. The caged compound remains stable for many hours in culture, but can be photochemically liberated rapidly and on demand with spatial control. Upon directed light exposure, the uncaged amino acid is available for local translation, enabling downstream proteomic interrogation via bioorthogonal conjugation. Exploiting the reactive azide moiety present on Ahas amino acid side chain, we demonstrate that newly synthesized proteins can be purified for quantitative proteomics or visualized in synthetic tissues with a new level of spatiotemporal control. Shedding light on when and where proteins are translated within living samples, we anticipate that laBONCAT will aid in understanding the progression of complex protein-related disorders.

Dynamic alterations of hepatocellular function by on-demand elasticity and roughness modulation

Temperature-responsive cell culture substrates reported here can be dynamically programmed to induce bulk softening and surface roughness changes in the presence of living cells. Alterations in hepatocellular function following temporally controlled substrate softening depend on the extent of stiff mechanical priming prior to user-induced material transition.

Introduction to Editorial Board Member: Professor Kristi S. Anseth

In this issue of Bioengineering and Translational Medicine, we are pleased to introduce our Editorial Board Member, Prof. Kristi S. Anseth. Prof. Anseth is a Distinguished Professor and the Tony Tisone Endowed Chair in Chemical and Biological Engineering, Associate Professor of Surgery, and the Associate Director of the BioFrontiers Institute at the University of Colorado Boulder. She was the first engineer to be named a Howard Hughes Medical Institute (HHMI) Investigator and is one of a select few individuals elected to all three United States National Academies (Sciences, Engineering, and Medicine) as well as to the National Academy of Inventors for her major contributions to the fields of biomaterials, tissue engineering, and regenerative medicine. Prof. Anseth earned her B.S. in Chemical Engineering with Highest Distinction from Purdue University where she performed undergraduate research in the laboratory of Prof. Nicholas Peppas. She completed her PhD in the Chemical Engineering department at the University of Colorado Boulder under the advisement of Prof. Christopher Bowman; her doctorate was completed in just 28 months and resulted in 10 first-author publications. Upon graduation, she assumed postdoctoral research at the Massachusetts Institute of Technology with Prof. Robert Langer. In 1996, after just 1 year away from Boulder, she returned to her doctoral alma mater as faculty in the Department of Chemical Engineering at the University of Colorado. Repurposing traditional polymer chemistry approaches for applications in the biological sciences, a theme that permeates much of her research, Prof. Anseths early efforts sought to synthesize and characterize biodegradable polymeric materials for controlled drug release.

Cyclic Stiffness Modulation of Cell-Laden Protein-Polymer Hydrogels in Response to User-Specified Stimuli Including Light

Although mechanical signals presented by the extracellular matrix are known to regulate many essential cell functions, the specific effects of these interactions, particularly in response to dynamic and heterogeneous cues, remain largely unknown. Here, a modular semisynthetic approach is introduced to create protein–polymer hydrogel biomaterials that undergo reversible stiffening in response to user‐specified inputs. Employing a novel dual‐chemoenzymatic modification strategy, fusion protein‐based gel crosslinkers are created that exhibit stimuli‐dependent intramolecular association. Linkers based on calmodulin yield calcium‐sensitive materials, while those containing the photosensitive light, oxygen, and voltage sensing domain 2 (LOV2) protein give phototunable constructs whose moduli can be cycled on demand with spatiotemporal control about living cells. These unique materials are exploited to demonstrate the significant role that cyclic mechanical loading plays on fibroblast‐to‐myofibroblast transdifferentiation in 3D space. The moduli‐switchable materials should prove useful for studies in mechanobiology, providing new avenues to probe and direct matrix‐driven changes in 4D cell physiology.

Polymer Design and Development

A growing interest to probe and direct stem cell fate has fueled the ongoing development of a wide variety of polymer-based platforms for inxa0vitro culture. Biomaterials with unique chemical and physical properties are afforded through careful selection of composition and can be engineered to mimic critical aspects of the native extracellular matrix. This chapter highlights recent progress in polymer design and development for 3D stem cell culture, comparing the advantages of both naturally and synthetic-based precursors. Special attention is given to natural materials derived from polysaccharides, peptides, and proteins, as well as smart synthetic polymer systems that exhibit responsiveness to environmental stimuli (e.g., electricity, temperature, enzyme, light, heat, pH).

Soft Shape-Memory Materials

Shape-memory systems represent an exciting class of “smart” materials that possess the unique capability to change from a temporary distorted structure back to a memorized permanent shape upon application of an external stimulus. Though metallic shape-memory alloys have gained traction as solid-state alternatives to conventional actuators in the automotive and robotics industry, shape-memory polymers (SMPs) are a cheap and efficient alternative with a diverse number of applications in the biomedical sector. Building on a variety of polymeric SMPs that offer one-way control over material geometry, exciting new research in reversible shape-memory systems has dramatically expanded the complexity over which researchers can dictate dynamic material properties. Such spatiotemporal control over material geometry will prove invaluable in the promising fields of drug delivery, tissue engineering, and regenerative medicine. In this chapter, we examine a variety of soft SMP and supramolecular biomaterial systems from the viewpoint of materials nanoarchitectonics.