Native tissues provide cells with complex, three dimensional (3D) environments comprised of hydrated networks of extracellular matrix proteins and sugars. By mimicking the dimensionality of native tissue while deconstructing the effects of environmental parameters, protein-based hydrogels serve as attractive, in vitro platforms to investigate cell-matrix interactions. For cell encapsulation, the process of hydrogel formation through physical or covalent crosslinking must be mild and cell compatible. While many chemical crosslinkers are commercially available for hydrogel formation, only a subset are cytocompatible; therefore, the identification of new and reliable cytocompatible crosslinkers allows for greater flexibility of hydrogel design for cell encapsulation applications. Here, we introduce tetrakis (hydroxymethyl) phosphonium chloride (THPC) as an inexpensive, amine-reactive, aqueous crosslinker for 3D cell encapsulation in protein-based hydrogels. We characterize the THPC-amine reaction by demonstrating its ability to react with primary and secondary amines of various amino acids. In addition, we demonstrate the utility of THPC to tune hydrogel gelation time (6.7 ± 0.2 to 27 ± 1.2 min) and mechanical properties (storage moduli ~250 Pa to ~2200 Pa) with a recombinant elastin-like protein. Lastly, we show cytocompatibility of THPC for cell encapsulation of two cell types, embryonic stem cells and neuronal cells, where cells exhibited the ability to differentiate and/or grow in elastin-like protein hydrogels.
Crosslinker; Hydrogel; Cell Encapsulation; Amine-reactive
biomaterials; micropatterning; skeletal muscle; tissue mimic; protein engineering
Systematically tunable in vitro platforms are invaluable in gaining insight to stem cell-microenvironment interactions in three-dimensional cultures. Utilizing recombinant protein technology, we independently tune hydrogel properties to systematically isolate the effects of matrix crosslinking density on cardiomyocyte differentiation, maturation, and function. We show that contracting human embryonic stem cell-derived cardiomyocytes (hESC-CMs) remain viable within four engineered elastin-like hydrogels of varying crosslinking densities with elastic moduli ranging from 0.45 to 2.4 kPa. Cardiomyocyte phenotype and function was maintained within hESC embryoid bodies for up to 2 weeks. Interestingly, increased crosslinking density was shown to transiently suspend spontaneous contractility. While encapsulated cells began spontaneous contractions at day 1 in hydrogels of the lowest crosslinking density, onset of contraction was increasingly delayed at higher crosslinking densities up to 6 days. However, once spontaneous contraction was restored, the rate of contraction was similar within all materials (71 ± 8 beats/min). Additionally, all groups successfully responded to electrical pacing at both 1 and 2 Hz. This study demonstrates that encapsulated hESC-CMs respond to 3D matrix crosslinking density within elastin-like hydrogels and stresses the importance of investigating temporal cellular responses in 3D cultures.
Photocrosslinkable, protein-engineered biomaterials combine a rapid, controllable, cytocompatible crosslinking method with a modular design strategy to create a new family of bioactive materials. These materials have a wide range of biomedical applications, including the development of bioactive implant coatings, drug delivery vehicles, and tissue engineering scaffolds. We present the successful functionalization of a bioactive elastin-like protein with photoreactive diazirine moieties. Scalable synthesis is achieved using a standard recombinant protein expression host followed by site-specific modification of lysine residues with a heterobifunctional N-hydroxysuccinimide ester-diazirine crosslinker. The resulting biomaterial is demonstrated to be processable by spin coating, drop casting, soft lithographic patterning, and mold casting to fabricate a variety of two- and three-dimensional photocrosslinked biomaterials with length scales spanning the nanometer to millimeter range. Protein thin films proved to be highly stable over a three-week period. Cell-adhesive functional domains incorporated into the engineered protein materials were shown to remain active post-photo-processing. Human adipose-derived stem cells achieved faster rates of cell adhesion and larger spread areas on thin films of the engineered protein compared to control substrates. The ease and scalability of material production, processing versatility, and modular bioactive functionality make this recombinantly engineered protein an ideal candidate for the development of novel biomaterial coatings, films, and scaffolds.
This study examined the homing capacity of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and their response to chemotactic gradients of stromal derived factor-1α (SDF). We have previously shown that EC derived from murine pluripotent stem cells can home to the ischemic hindlimb of the mouse. In the current study, we were interested to understand if ECs derived from human induced pluripotent stem cells are capable of homing. The homing capacity of iPSC-ECs was assessed after systemic delivery into immunodeficient mice with unilateral hindlimb ischemia. Furthermore, the iPSC-ECs were evaluated for their expression of CXCR4 and their ability to respond to SDF chemotactic gradients in vitro. Upon systemic delivery, the iPSC-ECs transiently localized to the lungs but did not home to the ischemic limb over the course of 14 days. To understand the mechanism of the lack of homing, the expression levels of the homing receptor, CXCR4, was examined at the transcriptional and protein levels. Furthermore, their ability to migrate in response to chemokines was assessed using microfluidic and scratch assays. Unlike ECs derived from syngeneic mouse pluripotent stem cells, human iPSC-ECs do not home to the ischemic mouse hindlimb. This lack of functional homing may represent an impairment of interspecies cellular communication or a difference in the differentiation state of the human iPSC-ECs. These results may have important implications in therapeutic delivery of iPSC-ECs.
Induced pluripotent stem cells; endothelial cells; CXCR4; SDF-1; homing; hindlimb ischemia
The native stem cell niche is a dynamic and complex microenvironment. Recapitulating this niche is a critical focus within the fields of stem cell biology, tissue engineering, and regenerative medicine and requires the development of well-defined, tunable materials. Recent biomaterial design strategies seek to create engineered matrices that interact with cells at the molecular scale and allow on-demand, cell-triggered matrix modifications. Peptide and protein engineering can accomplish these goals through the molecular-level design of bioinductive and bioresponsive materials. This brief review focuses on engineered peptide and protein materials suitable for use as in vitro neural stem cell niche mimics and in vivo central nervous system repair. A key hallmark of these materials is the immense design freedom to specify the exact amino acid sequence leading to multi-functional bulk materials with tunable properties. These advanced materials are engineered using rational design strategies to recapitulate key aspects of the native neural stem cell niche. The resulting materials often combine the advantages of biological matrices with the engineering control of synthetic polymers. Future design strategies are expected to endow these materials with multiple layers of bi-directional feedback between the cell and the matrix, which will lead to more advanced mimics of the highly dynamic neural stem cell niche.
Engineered peptide; Engineered protein; Tissue engineering; Neural stem cell; Stem cell niche; Extracellular matrix
Predictable tuning of bulk mechanics from the molecular level remains elusive in many physical hydrogel systems due to the reliance on non-specific and non-stoichiometric chain interactions for network formation. We describe a Mixing-Induced Two-Component Hydrogel (MITCH) system, in which network assembly is driven by specific and stoichiometric peptide-peptide binding interactions. By integrating protein science methodologies with simple polymer physics model, we manipulate the polypeptide binding interactions and demonstrate the direct ability to predict the resulting effects on network crosslinking density, sol-gel phase behavior, and gel mechanics.
physical hydrogels; protein engineering; biomaterials
Traditional materials used as in vitro cell culture substrates are rigid and flat surfaces that lack the exquisite nano- and micro-scale features of the in vivo extracellular environment. While these surfaces can be coated with harvested extracellular matrix (ECM) proteins to partially recapitulate the bio-instructive nature of the ECM, these harvested proteins often exhibit large batch-to-batch variability and can be difficult to customize for specific biological studies. In contrast, recombinant protein technology can be utilized to synthesize families of protein-engineered biomaterials that are cyto-compatible, reproducible, and fully customizable.
Scope of Review
Here we describe a modular design strategy to synthesize protein-engineered biomaterials that fuse together multiple repeats of nanoscale peptide design motifs into full-length engineered ECM mimetics.
Due to the molecular-level precision of recombinant protein synthesis, these biomaterials can be tailored to include a variety of bio-instructional ligands at specified densities, to exhibit mechanical properties that match those of native tissue, and to include proteolytic target sites that enable cell-triggered scaffold remodeling. Furthermore, these biomaterials can be processed into forms that are injectable for minimally-invasive delivery or spatially patterned to enable the release of multiple drugs with distinct release kinetics.
Given the reproducibility and flexibility of these protein-engineered biomaterials, they are ideal substrates for reductionist biological studies of cell-matrix interactions, for in vitro models of physiological processes, and for bio-instructive scaffolds in regenerative medicine therapies.
biomaterial; protein engineering; stem cell niche; extracellular matrix; tissue engineering; regenerative medicine
Cell transplantation is a promising therapy for a myriad of debilitating diseases; however, current delivery protocols using direct injection result in poor cell viability. We demonstrate that during the actual cell injection process, mechanical membrane disruption results in significant acute loss of viability at clinically relevant injection rates. As a strategy to protect cells from these damaging forces, we hypothesize that cell encapsulation within hydrogels of specific mechanical properties will significantly improve viability. We use a controlled in vitro model of cell injection to demonstrate success of this acute protection strategy for a wide range of cell types including human umbilical vein endothelial cells (HUVEC), human adipose stem cells, rat mesenchymal stem cells, and mouse neural progenitor cells. Specifically, alginate hydrogels with plateau storage moduli (G′) ranging from 0.33 to 58.1 Pa were studied. A compliant crosslinked alginate hydrogel (G′=29.6 Pa) yielded the highest HUVEC viability, 88.9%±5.0%, while Newtonian solutions (i.e., buffer only) resulted in 58.7%±8.1% viability. Either increasing or decreasing the hydrogel storage modulus reduced this protective effect. Further, cells within noncrosslinked alginate solutions had viabilities lower than media alone, demonstrating that the protective effects are specifically a result of mechanical gelation and not the biochemistry of alginate. Experimental and theoretical data suggest that extensional flow at the entrance of the syringe needle is the main cause of acute cell death. These results provide mechanistic insight into the role of mechanical forces during cell delivery and support the use of protective hydrogels in future clinical stem cell injection studies.
A critical property of biomaterials for use in regenerative medicine applications is the ability to promote angiogenesis, the formation of new vascular networks, to support regenerating tissues. Recent studies have demonstrated that a complex interplay exists between biomechanical and biochemical regulators of endothelial cell sprouting, an early step in angiogenesis. Here, we use a microfluidic platform to study the pathfinding behaviors induced by various stable vascular endothelial growth factor (VEGF) gradients during sprouting morphogenesis within biomaterials. Quantitative, time-lapse analysis of endothelial sprouting demonstrated that the ability of VEGF to regulate sprout orientation during several stages of sprouting morphogenesis (initiation, elongation, and turning navigation) was biomaterial dependent. Identical VEGF gradients induced different types of coordinated cell movements depending on the density of the surrounding collagen/fibronectin matrix. In denser matrices, sprouts were more likely to have an initial orientation aligned parallel to the VEGF gradient. In contrast, in less dense matrices, sprouts were more likely to initially misalign with the VEGF gradient; however, these sprouts underwent significant turning and navigation to eventually reorient to be parallel to the VEGF gradient. These less dense matrices required shallower VEGF gradients and demonstrated lower activating VEGF thresholds to induce proper sprout alignment and pathfinding. These results encourage the future use of microfluidic platforms to probe fundamental aspects of matrix effects on angiogenesis, to screen biomaterials for angiogenic potential, and to design ex vivo tissues with aligned vascular networks.
The orphan G protein-coupled receptor GPR124/TEM5 is highly expressed in central nervous system (CNS) endothelium. Here, complete null or endothelial-specific GPR124 deletion produced embryonic lethality from CNS-specific angiogenesis arrest in forebrain and neural tube. Conversely, GPR124 overexpression throughout all adult vascular beds produced CNS-specific hyperproliferative vascular malformations. In vivo, GPR124 functioned cell-autonomously in endothelium to regulate sprouting, migration, and developmental expression of the blood-brain barrier marker Glut1, while in vitro, GPR124 mediated Cdc42-dependent directional migration to forebrain-derived, VEGF-independent cues. Our results demonstrate CNS-specific angiogenesis regulation by an endothelial receptor, and illuminate functions of the poorly understood adhesion GPCR subfamily. Further, the striking functional tropism of GPR124 marks this receptor as a therapeutic target for CNS-related vascular pathologies.
Optical observations of 100 nm metallic magnetic nanoparticles are used to study their magnetic field induced self assembly. Chains with lengths of tens of microns are observed to form within minutes at nanoparticle concentrations of 1010 per mL. Chain rotation and magnetophoresis are readily observed, and SEM reveals that long chains are not simple single particle filaments. Similar chains are detected for several 100 nm commercial bio-separation nanoparticles. We demonstrate the staged magnetic condensation of different types of nanoparticles into composite structures and show that magnetic chains bind to immunomagnetically labeled cells, serving as temporary handles which allow novel magnetic cell manipulations.
magnetic nanoparticles; nanoparticle characterization; biomedical applications; bimetallic nanoparticle; magnetic properties
The highly debilitating nature of spinal cord injuries has provided much inspiration for the design of novel biomaterials that can stimulate cellular regeneration and functional recovery. Many experts agree that the greatest hope for treatment of spinal cord injuries will involve a combinatorial approach that integrates biomaterial scaffolds, cell transplantation, and molecule delivery. This manuscript presents a comprehensive review of biomaterial-scaffold design strategies currently being applied to the development of nerve guidance channels and hydrogels that more effectively stimulate spinal cord tissue regeneration. To enhance the regenerative capacity of these two scaffold types, researchers are focusing on optimizing the mechanical properties, cell-adhesivity, biodegradability, electrical activity, and topography of synthetic and natural materials, and are developing mechanisms to use these scaffolds to deliver cells and biomolecules. Developing scaffolds that address several of these key design parameters will lead to more successful therapies for the regeneration of spinal cord tissue.
biomaterials; peripheral nerve injury; regeneration; spinal cord injury; therapeutic approaches for the treatment of CNS injury
Spatial patterning of proteins is a valuable technique for many biological applications and is the prevailing tool for defining microenvironments for cells in culture, a required procedure in developmental biology and tissue engineering research. However, it is still challenging to achieve protein patterns that closely mimic native microenvironments, such as gradient protein distributions with desirable mechanical properties. By combining projection dynamic mask lithography and protein engineering with non-canonical photosensitive amino acids, we demonstrate a simple, scalable strategy to fabricate any user-defined 2D or 3D stable gradient pattern with complex geometries from an artificial extracellular matrix (aECM) protein. We show that the elastic modulus and chemical nature of the gradient profile are biocompatible and allow useful applications in cell biological research.
Protein patterning; Protein lithography; Protein gradient; DMD (digital micromirror device); Protein engineering; Non-canonical amino acids
Neural prosthetic implants are currently being developed for the treatment and study of both peripheral and central nervous system disorders. Effective integration of these devices upon implantation is a critical hurdle to achieving function. As a result, much attention has been directed towards the development of biocompatible coatings that prolong their in vivo lifespan. In this work, we present a novel approach to fabricate such coatings, which specifically involves the use of surface-adsorbed, nanoscale-designed protein polymers to prepare reproducible, customized surfaces. A nanoscale modular design strategy was employed to synthesize six engineered, recombinant proteins intended to mimic aspects of the extracellular matrix proteins fibronectin, laminin, and elastin as well as the cell–cell adhesive protein neural cell adhesion molecule. Physical adsorption isotherms were experimentally determined for these engineered proteins, allowing for direct calculation of the available ligand density present on coated surfaces. As confirmation that ligand density in these engineered systems impacts neuronal cell behavior, we demonstrate that increasing the density of fibronectin-derived RGD ligands on coated surfaces while maintaining uniform protein surface coverage results in enhanced neurite extension of PC-12 cells. Therefore, this engineered protein adsorption approach allows for the facile preparation of tunable, quantifiable, and reproducible surfaces for in vitro studies of cell–ligand interactions and for potential application as coatings on neural implants.
biocompatible; protein; adsorption; coatings; implants; neural; elastin