Co-reporter:Huiyuan Wang;Danqing Zhu;Alexra Paul;Lei Cai;Annika Enejder;Fan Yang
Advanced Functional Materials 2017 Volume 27(Issue 28) pp:
Publication Date(Web):2017/07/01
DOI:10.1002/adfm.201605609
Shear-thinning, self-healing hydrogels are promising vehicles for therapeutic cargo delivery due to their ability to be injected using minimally invasive surgical procedures. An injectable hydrogel using a novel combination of dynamic covalent crosslinking with thermoresponsive engineered proteins is presented. Ex situ at room temperature, rapid gelation occurs through dynamic covalent hydrazone bonds by simply mixing two components: hydrazine-modified elastin-like protein (ELP) and aldehyde-modified hyaluronic acid. This hydrogel provides significant mechanical protection to encapsulated human mesenchymal stem cells during syringe needle injection and rapidly recovers after injection to retain the cells homogeneously within a 3D environment. In situ, the ELP undergoes a thermal phase transition, as confirmed by coherent anti-Stokes Raman scattering microscopy observation of dense ELP thermal aggregates. The formation of the secondary network reinforces the hydrogel and results in a tenfold slower erosion rate compared to a control hydrogel without secondary thermal crosslinking. This improved structural integrity enables cell culture for three weeks postinjection, and encapsulated cells maintain their ability to differentiate into multiple lineages, including chondrogenic, adipogenic, and osteogenic cell types. Together, these data demonstrate the promising potential of ELP–HA hydrogels for injectable stem cell transplantation and tissue regeneration.
Co-reporter:Rebecca L. DiMarco, Daniel R. Hunt, Ruby E. Dewi, Sarah C. Heilshorn
Biomaterials 2017 Volume 129(Volume 129) pp:
Publication Date(Web):1 June 2017
DOI:10.1016/j.biomaterials.2017.03.023
The Caco-2 assay has achieved wide popularity among pharmaceutical companies in the past two decades as an in vitro method for estimation of in vivo oral bioavailability of pharmaceutical compounds during preclinical characterization. Despite its popularity, this assay suffers from a severe underprediction of the transport of drugs which are absorbed paracellularly, that is, which pass through the cell-cell tight junctions of the absorptive cells of the small intestine. Here, we propose that simply replacing the collagen I matrix employed in the standard Caco-2 assay with an engineered matrix, we can control cell morphology and hence regulate the cell-cell junctions that dictate paracellular transport. Specifically, we use a biomimetic engineered extracellular matrix (eECM) that contains modular protein domains derived from two ECM proteins found in the small intestine, fibronectin and elastin. This eECM allows us to independently tune the density of cell-adhesive RGD ligands presented to Caco-2 cells as well as the mechanical stiffness of the eECM. We observe that lower amounts of RGD ligand presentation as well as decreased matrix stiffness results in Caco-2 morphologies that more closely resemble primary small intestinal epithelial cells than Caco-2 cells cultured on collagen. Additionally, these matrices result in Caco-2 monolayers with decreased recruitment of actin to the apical junctional complex and increased expression of claudin-2, a tight junction protein associated with higher paracellular permeability that is highly expressed throughout the small intestine. Consistent with these morphological differences, drugs known to be paracellularly transported in vivo exhibited significantly improved transport rates in this modified Caco-2 model. As expected, permeability of transcellularly transported drugs remained unaffected. Thus, we have demonstrated a method of improving the physiological accuracy of the Caco-2 assay that could be readily adopted by pharmaceutical companies without major changes to their current testing protocols.
Co-reporter:Christopher M. Madl;Lily M. Katz
Advanced Functional Materials 2016 Volume 26( Issue 21) pp:3612-3620
Publication Date(Web):
DOI:10.1002/adfm.201505329
Covalently-crosslinked hydrogels are commonly used as 3D matrices for cell culture and transplantation. However, the crosslinking chemistries used to prepare these gels generally cross-react with functional groups present on the cell surface, potentially leading to cytotoxicity and other undesired effects. Bio-orthogonal chemistries have been developed that do not react with biologically relevant functional groups, thereby preventing these undesirable side reactions. However, previously developed biomaterials using these chemistries still possess less than ideal properties for cell encapsulation, such as slow gelation kinetics and limited tuning of matrix mechanics and biochemistry. Here, engineered elastin-like proteins (ELPs) are developed that crosslink via strain-promoted azide-alkyne cycloaddition (SPAAC) or Staudinger ligation. The SPAAC-crosslinked materials form gels within seconds and complete gelation within minutes. These hydrogels support the encapsulation and phenotypic maintenance of human mesenchymal stem cells, human umbilical vein endothelial cells, and murine neural progenitor cells. SPAAC-ELP gels exhibit independent tuning of stiffness and cell adhesion, with significantly improved cell viability and spreading observed in materials containing a fibronectin-derived arginine-glycine-aspartic acid (RGD) domain. The crosslinking chemistry used permits further material functionalization, even in the presence of cells and serum. These hydrogels are anticipated to be useful in a wide range of applications, including therapeutic cell delivery and bioprinting.
Co-reporter:Patrick L. Benitez, Shamik Mascharak, Amy C. Proctor and Sarah C. Heilshorn
Integrative Biology 2016 vol. 8(Issue 1) pp:50-61
Publication Date(Web):11 Dec 2015
DOI:10.1039/C5IB00258C
While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to the design of implantable biomaterials because the local and global ligand densities that enable clustering-enhanced integrin signaling were unpredictable. Here, two general design principles for biomaterial ligand clustering are elucidated. First, clustering ligands enhances integrin-dependent signals when the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand’s effective dissociation constant (KD,eff). Second, clustering ligands enhances integrin activation when the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. To identify these principles, we fabricated a series of elastin-like, electrospun fabrics with independent control over the local (0 to 122000 ligands μm−2) and global (0 to 71000 ligand μm−2) densities of an arginine–glycine–aspartate (RGD) ligand. Antibody blocking studies confirmed that human umbilical vein endothelial cell adhesion to these protein-engineered biomaterials was primarily due to αVβ3 integrin binding. Clustering ligands enhanced cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand's KD,eff of 12000 RGD μm−2. Near this global ligand density, cells on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this enhanced ligand-clustering effect was not observed above a threshold cut-off concentration. At a local ligand density of 122000 RGD μm−2, cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer able to effectively engage with their target ligands. Together, these two insights into the cellular responses to ligand clustering at the cell–matrix interface may serve as design principles when developing future generations of implantable biomaterials.
Co-reporter:Jordan Raphel, Mark Holodniy, Stuart B. Goodman, Sarah C. Heilshorn
Biomaterials 2016 84() pp: 301-314
Publication Date(Web):April 2016
DOI:10.1016/j.biomaterials.2016.01.016
The two leading causes of failure for joint arthroplasty prostheses are aseptic loosening and periprosthetic joint infection. With the number of primary and revision joint replacement surgeries on the rise, strategies to mitigate these failure modes have become increasingly important. Much of the recent work in this field has focused on the design of coatings either to prevent infection while ignoring bone mineralization or vice versa, to promote osseointegration while ignoring microbial susceptibility. However, both coating functions are required to achieve long-term success of the implant; therefore, these two modalities must be evaluated in parallel during the development of new orthopaedic coating strategies. In this review, we discuss recent progress and future directions for the design of multifunctional orthopaedic coatings that can inhibit microbial cells while still promoting osseointegration.
Co-reporter:Matthew G. Haugh, Sarah C. Heilshorn
Current Opinion in Solid State and Materials Science 2016 Volume 20(Issue 4) pp:171-179
Publication Date(Web):August 2016
DOI:10.1016/j.cossms.2016.04.001
•Ligand chemistry and substrate mechanics are integrated to define the mechanical resistance presented to MSCs.•Distinctive changes in mechanics moving from 2D to 3D macro-porous or non-macro-porous substrates.•Macro-porous substrates present a heterogeneous mechanical environment.•The specificity of integrin–ligand interactions is altered within non-macro-porous substrates.•Mechanotransduction within covalently crosslinked, non-macro-porous substrates requires degradation.The role of substrate mechanics in guiding mesenchymal stem cell (MSC) fate has been the focus of much research over the last decade. More recently, the complex interplay between substrate mechanics and other material properties such as ligand chemistry and substrate degradability to regulate MSC differentiation has begun to be elucidated. Additionally, there are several changes in the presentation of these material properties as the dimensionality is altered from two- to three-dimensional substrates, which may fundamentally alter our understanding of substrate-induced mechanotransduction processes. In this review, an overview of recent findings that highlight the material properties that are important in guiding MSC fate decisions is presented, with a focus on underlining gaps in our existing knowledge and proposing potential directions for future research.
Co-reporter:Huiyuan Wang
Advanced Materials 2015 Volume 27( Issue 25) pp:3717-3736
Publication Date(Web):
DOI:10.1002/adma.201501558
Adaptable hydrogels have recently emerged as a promising platform for three-dimensional (3D) cell encapsulation and culture. In conventional, covalently crosslinked hydrogels, degradation is typically required to allow complex cellular functions to occur, leading to bulk material degradation. In contrast, adaptable hydrogels are formed by reversible crosslinks. Through breaking and re-formation of the reversible linkages, adaptable hydrogels can be locally modified to permit complex cellular functions while maintaining their long-term integrity. In addition, these adaptable materials can have biomimetic viscoelastic properties that make them well suited for several biotechnology and medical applications. In this review, an overview of adaptable-hydrogel design considerations and linkage selections is presented, with a focus on various cell-compatible crosslinking mechanisms that can be exploited to form adaptable hydrogels for tissue engineering.
Co-reporter:Lei Cai;Ruby E. Dewi
Advanced Functional Materials 2015 Volume 25( Issue 9) pp:1344-1351
Publication Date(Web):
DOI:10.1002/adfm.201403631
Stem cell transplantation via direct injection is a minimally invasive strategy being explored for treatment of a variety of injuries and diseases. Injectable hydrogels with shear moduli <50 Pa can mechanically protect cells during the injection process; however, these weak gels typically biodegrade within 1–2 weeks, which may be too fast for many therapeutic applications. To address this limitation, an injectable hydrogel is designed that undergoes two different physical crosslinking mechanisms. The first crosslinking step occurs ex vivo through peptide-based molecular recognition to encapsulate cells within a weak gel that provides mechanical protection from injection forces. The second crosslinking step occurs in situ to form a reinforcing network that significantly retards material biodegradation and prolongs cell retention time. Human adipose-derived stem cells are transplanted into the subcutaneous space of a murine model using hand-injection through a 28-gauge syringe needle. Cells delivered within the double-network hydrogel are significantly protected from mechanical damage and have significantly enhanced in vivo cell retention rates compared to delivery within saline and single network hydrogels. These results demonstrate that in situ formation of a reinforcing network within an already existing hydrogel can greatly improve transplanted cell retention, thereby enhancing potential regenerative medicine therapies.
Co-reporter:Nicole H. Romano, Christopher M. Madl, Sarah C. Heilshorn
Acta Biomaterialia 2015 Volume 11() pp:48-57
Publication Date(Web):1 January 2015
DOI:10.1016/j.actbio.2014.10.008
Abstract
The innate biological response to peripheral nerve injury involves a complex interplay of multiple molecular cues to guide neurites across the injury gap. Many current strategies to stimulate regeneration take inspiration from this biological response. However, little is known about the balance of cell–matrix and Schwann cell–neurite dynamics required for regeneration of neural architectures. We present an engineered extracellular matrix (eECM) microenvironment with tailored cell–matrix and cell–cell interactions to study their individual and combined effects on neurite outgrowth. This eECM regulates cell–matrix interactions by presenting integrin-binding RGD (Arg–Gly–Asp) ligands at specified densities. Simultaneously, the addition or exclusion of nerve growth factor (NGF) is used to modulate L1CAM-mediated Schwann cell–neurite interactions. Individually, increasing the RGD ligand density from 0.16 to 3.2 mM resulted in increasing neurite lengths. In matrices presenting higher RGD ligand densities, neurite outgrowth was synergistically enhanced in the presence of soluble NGF. Analysis of Schwann cell migration and co-localization with neurites revealed that NGF enhanced cooperative outgrowth between the two cell types. Interestingly, neurites in NGF-supplemented conditions were unable to extend on the surrounding eECM without the assistance of Schwann cells. Blocking studies revealed that L1CAM is primarily responsible for these Schwann cell–neurite interactions. Without NGF supplementation, neurite outgrowth was unaffected by L1CAM blocking or the depletion of Schwann cells. These results underscore the synergistic interplay between cell–matrix and cell–cell interactions in enhancing neurite outgrowth for peripheral nerve regeneration.
Co-reporter:Rebecca L. DiMarco, Ruby E. Dewi, Gabriela Bernal, Calvin Kuo and Sarah C. Heilshorn
Biomaterials Science 2015 vol. 3(Issue 10) pp:1376-1385
Publication Date(Web):16 Jul 2015
DOI:10.1039/C5BM00108K
Though in vitro culture of primary intestinal organoids has gained significant momentum in recent years, little has been done to investigate the impact of microenvironmental cues provided by the encapsulating matrix on the growth and development of these fragile cultures. In this work, the impact of various in vitro culture parameters on primary adult murine organoid formation and growth are analyzed with a focus on matrix properties and geometric culture configuration. The air–liquid interface culture configuration was found to result in enhanced organoid formation relative to a traditional submerged configuration. Additionally, through use of a recombinantly engineered extracellular matrix (eECM), the effects of biochemical and biomechanical cues were independently studied. Decreasing mechanical stiffness and increasing cell adhesivity were found to increase organoid yield. Tuning of eECM properties was used to obtain organoid formation efficiency values identical to those observed in naturally harvested collagen I matrices but within a stiffer construct with improved ease of physical manipulation. Increased ability to remodel the surrounding matrix through mechanical or enzymatic means was also shown to enhance organoid formation. As the engineering and tunability of recombinant matrices is essentially limitless, continued property optimization may result in further improved matrix performance and may help to identify additional microenvironmental cues that directly impact organoid formation, development, differentiation, and functional behavior. Continued culture of primary organoids in recombinant matrices could therefore prove to be largely advantageous in the field of intestinal tissue engineering for applications in regenerative medicine and in vitro tissue mimics.
Co-reporter:Meghaan M. Ferreira, Ruby E. Dewi and Sarah C. Heilshorn
Integrative Biology 2015 vol. 7(Issue 5) pp:569-579
Publication Date(Web):14 Apr 2015
DOI:10.1039/C5IB00060B
Exposing myoblasts to basic fibroblast growth factor (bFGF), which is released after muscle injury, results in receptor phosphorylation, faster migration, and increased proliferation. These effects occur on time scales that extend across three orders of magnitude (100–103 minutes). Finite element modeling of Transwell assays, which are traditionally used to assess chemotaxis, revealed that the bFGF gradient formed across the membrane pore is short-lived and diminishes 45% within the first minute. Thus, to evaluate bFGF-induced migration over 102 minutes, we employed a microfluidic assay capable of producing a stable, linear concentration gradient to perform single-cell analyses of chemokinesis and chemotaxis. We hypothesized that the composition of the underlying extracellular matrix (ECM) may affect the behavioral response of myoblasts to soluble bFGF, as previous work with other cell types has suggested crosstalk between integrin and fibroblast growth factor (FGF) receptors. Consistent with this notion, we found that bFGF significantly reduced the doubling time of myoblasts cultured on laminin but not fibronectin or collagen. Laminin also promoted significantly faster migration speeds (13.4 μm h−1) than either fibronectin (10.6 μm h−1) or collagen (7.6 μm h−1) without bFGF stimulation. Chemokinesis driven by bFGF further increased migration speed in a strictly additive manner, resulting in an average increase of 2.3 μm h−1 across all ECMs tested. We observed relatively mild chemoattraction (∼67% of myoblast population) in response to bFGF gradients of 3.2 ng mL−1 mm−1 regardless of ECM identity. Thus, while ECM-bFGF crosstalk did impact chemoproliferation, it did not have a significant effect on chemokinesis or chemotaxis. These data suggest that the main physiological effect of bFGF on myoblast migration is chemokinesis and that changes in the surrounding ECM, resulting from aging and/or disease may impact muscle regeneration by altering myoblast migration and proliferation.
Co-reporter:Kelly N. L. Huggins;Alia P. Schoen;Manickam Adhimoolam Arunagirinathan
Advanced Functional Materials 2014 Volume 24( Issue 48) pp:7737-7744
Publication Date(Web):
DOI:10.1002/adfm.201402049
The use of biological scaffolds to template inorganic material offers a strategy to synthesize precise composite nanostructures of different sizes and shapes. Proteins are unique biological scaffolds that consist of multiple binding regions or epitope sites that site-specifically associate with conserved amino acid sequences within protein-binding partners. These binding regions can be exploited as synthesis sites for multiple inorganic species within the same protein scaffold, resulting in bimetallic inorganic nanostructures. This strategy is demonstrated with the scaffold protein clathrin, which self-assembles into spherical cages. Specifically, tether peptides that noncovalently associate with distinct clathrin epitope sites, while initiating simultaneous synthesis of two inorganic species within the assembled clathrin protein cage, are designed. The flexibility and diversity of this unique biotemplating strategy is demonstrated by synthesizing two types of composite structures (silver–gold mixed bimetallic and silver–gold core–shell nanostructures) from a single clathrin template. This noncovalent, Template Engineering Through Epitope Recognition, or TEThER, strategy can be readily applied to any protein system with known epitope sites to template a variety of bimetallic structures without the need for chemical or genetic mutations.
Co-reporter:Widya Mulyasasmita, Lei Cai, Ruby E. Dewi, Arshi Jha, Sabrina D. Ullmann, Richard H. Luong, Ngan F. Huang, Sarah C. Heilshorn
Journal of Controlled Release 2014 Volume 191() pp:71-81
Publication Date(Web):10 October 2014
DOI:10.1016/j.jconrel.2014.05.015
To translate recent advances in induced pluripotent stem cell biology to clinical regenerative medicine therapies, new strategies to control the co-delivery of cells and growth factors are needed. Building on our previous work designing Mixing-Induced Two-Component Hydrogels (MITCHs) from engineered proteins, here we develop protein–polyethylene glycol (PEG) hybrid hydrogels, MITCH-PEG, which form physical gels upon mixing for cell and growth factor co-delivery. MITCH-PEG is a mixture of C7, which is a linear, engineered protein containing seven repeats of the CC43 WW peptide domain (C), and 8-arm star-shaped PEG conjugated with either one or two repeats of a proline-rich peptide to each arm (P1 or P2, respectively). Both 20 kDa and 40 kDa star-shaped PEG variants were investigated, and all four PEG-peptide variants were able to undergo a sol–gel phase transition when mixed with the linear C7 protein at constant physiological conditions due to noncovalent hetero-dimerization between the C and P domains. Due to the dynamic nature of the C–P physical crosslinks, all four gels were observed to be reversibly shear-thinning and self-healing. The P2 variants exhibited higher storage moduli than the P1 variants, demonstrating the ability to tune the hydrogel bulk properties through a biomimetic peptide-avidity strategy. The 20 kDa PEG variants exhibited slower release of encapsulated vascular endothelial growth factor (VEGF), due to a decrease in hydrogel mesh size relative to the 40 kDa variants. Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) adopted a well-spread morphology within three-dimensional MITCH-PEG cultures, and MITCH-PEG provided significant protection from cell damage during ejection through a fine-gauge syringe needle. In a mouse hindlimb ischemia model of peripheral arterial disease, MITCH-PEG co-delivery of hiPSC-ECs and VEGF was found to reduce inflammation and promote muscle tissue regeneration compared to a saline control.A biomimetic avidity strategy was used to design a family of Mixing-Induced Two-Component Hydrogels for the injectable co-delivery of growth factors and human induced pluripotent stem cell-derived endothelial cells.
Co-reporter:Hui Xu, Meghaan M. Ferreira and Sarah C. Heilshorn
Lab on a Chip 2014 vol. 14(Issue 12) pp:2047-2056
Publication Date(Web):14 Apr 2014
DOI:10.1039/C4LC00162A
A deep understanding of the mechanisms behind neurite polarization and axon path-finding is important for interpreting how the human body guides neurite growth during development and response to injury. Further, it is of great clinical importance to identify diffusible chemical cues that promote neurite regeneration for nervous tissue repair. Despite the fast development of various types of concentration gradient generators, it has been challenging to fabricate neuron-friendly (i.e. shear-free and biocompatible for neuron growth and maturation) devices to create stable gradients, particularly for fast diffusing small molecules, which typically require high flow and shear rates. Here we present a finite element analysis for a polydimethylsiloxane/polyethylene glycol diacrylate (PDMS/PEG-DA) based gradient generator, describe the microfabrication process, and validate its use for neuronal axon polarization studies. This device provides a totally shear-free, biocompatible microenvironment with a linear and stable concentration gradient of small molecules such as forskolin. The gradient profile in this device can be customized by changing the composition or width of the PEG-DA barriers during direct UV photo-patterning within a permanently bonded PDMS device. Primary rat cortical neurons (embryonic E18) exposed to soluble forskolin gradients for 72 h exhibited statistically significant polarization and guidance of their axons. This device provides a useful platform for both chemotaxis and directional guidance studies, particularly for shear sensitive and non-adhesive cell cultures, while allowing fast new device design prototyping at a low cost.
Co-reporter:Lei Cai, Sarah C. Heilshorn
Acta Biomaterialia 2014 Volume 10(Issue 4) pp:1751-1760
Publication Date(Web):April 2014
DOI:10.1016/j.actbio.2013.12.028
Abstract
The natural extracellular matrix (ECM), with its multitude of evolved cell-instructive and cell-responsive properties, provides inspiration and guidelines for the design of engineered biomaterials. One strategy to create ECM-mimetic materials is the modular design of protein-based engineered ECM (eECM) scaffolds. This modular design strategy involves combining multiple protein domains with different functionalities into a single, modular polymer sequence, resulting in a multifunctional matrix with independent tunability of the individual domain functions. These eECMs often enable decoupled control over multiple material properties for fundamental studies of cell–matrix interactions. In addition, since the eECMs are frequently composed entirely of bioresorbable amino acids, these matrices have immense clinical potential for a variety of regenerative medicine applications. This brief review demonstrates how fundamental knowledge gained from structure–function studies of native proteins can be exploited in the design of novel protein-engineered biomaterials. While the field of protein-engineered biomaterials has existed for over 20 years, the community is only now beginning to fully explore the diversity of functional peptide modules that can be incorporated into these materials. We have chosen to highlight recent examples that either (i) demonstrate exemplary use as matrices with cell-instructive and cell-responsive properties or (ii) demonstrate outstanding creativity in terms of novel molecular-level design and macro-level functionality.
Co-reporter:Thomas H. Barker, Sarah C. Heilshorn
Acta Biomaterialia 2014 Volume 10(Issue 4) pp:1487
Publication Date(Web):April 2014
DOI:10.1016/j.actbio.2014.02.001
Co-reporter:Midori Greenwood-Goodwin, Eric S. Teasley and Sarah C. Heilshorn
Biomaterials Science 2014 vol. 2(Issue 11) pp:1627-1639
Publication Date(Web):07 Aug 2014
DOI:10.1039/C4BM00142G
Engineered biomimetic microenvironments from hydrogels are an emerging strategy to achieve lineage-specific differentiation in vitro. In addition to recapitulating critical matrix cues found in the native three-dimensional (3D) niche, the hydrogel can also be designed to deliver soluble factors that are present within the native inductive microenvironment. We demonstrate a versatile materials approach for the dual-stage delivery of multiple soluble factors within a 3D hydrogel to induce adipogenesis. We use a mixing-induced two-component hydrogel (MITCH) embedded with alginate microgels to deliver two pro-adipogenic soluble factors, fibroblast growth factor 1 (FGF-1) and bone morphogenetic protein 4 (BMP-4) with two distinct delivery profiles. We show that dual-stage delivery of FGF-1 and BMP-4 to human adipose-derived stromal cells (hADSCs) significantly increases lipid accumulation compared with the simultaneous delivery of both growth factors together. Furthermore, dual-stage growth factor delivery within a 3D hydrogel resulted in substantially more lipid accumulation compared to identical delivery profiles in 2D cultures. Gene expression analysis shows upregulation of key adipogenic markers indicative of brown-like adipocytes. These data suggest that dual-stage release of FGF-1 and BMP-4 within 3D microenvironments can promote the in vitro development of mature adipocytes.
Co-reporter:Lei Cai, Cong B. Dinh and Sarah C. Heilshorn
Biomaterials Science 2014 vol. 2(Issue 5) pp:757-765
Publication Date(Web):04 Feb 2014
DOI:10.1039/C3BM60293A
Immobilization of growth factors to polymeric matrices has been a common strategy in the design of tissue engineering scaffolds to promote tissue regeneration, which requires complex cell signaling events with the surrounding matrix. However, the use of large protein growth factors in polymeric scaffolds is often plagued by immunogenicity, short in vivo half-lives, and reduced bioactivity. To address these concerns, we developed a single-step, cell-compatible strategy to tether small, growth-factor-mimetic peptides into a protein-engineered hydrogel with tunable biomaterial properties. Specifically, we covalently immobilized the QK peptide, an angiogenic peptide mimicking the receptor-binding region of vascular endothelial growth factor (VEGF), within tunable elastin-like polypeptide (ELP) hydrogels that include a cell-adhesive RGD sequence. Using a cell-compatible, amine-reactive crosslinker, we conducted a one-pot synthesis to simultaneously encapsulate cells while precisely controlling the QK grafting density (10 nM–100 μM) in the ELP hydrogels without altering other material properties. Fluorescence analysis of fluor-labeled QK peptides demonstrated that the conjugation efficiency to ELP hydrogels was >75% and that covalent immobilization effectively eliminates all QK diffusion. Compared with pristine ELP hydrogels, human umbilical vein endothelial cell (HUVEC) proliferation was significantly enhanced on ELP hydrogels immobilized with 10 nM or 1 μM QK. Moreover, upon encapsulation within tethered QK-ELP hydrogels, HUVEC spheroids maintained near 100% viability and demonstrated significantly more three-dimensional outgrowth compared to those supplemented with soluble QK peptide at the same concentration. These results encourage the further development of protein-engineered scaffolds decorated with growth-factor-mimetic peptides to provide long-term biological signals using this versatile, single-step synthesis.
Co-reporter:Rebecca L. DiMarco, James Su, Kelley S. Yan, Ruby Dewi, Calvin J. Kuo and Sarah C. Heilshorn
Integrative Biology 2014 vol. 6(Issue 2) pp:127-142
Publication Date(Web):28 Nov 2013
DOI:10.1039/C3IB40188J
Multiple culture techniques now exist for the long-term maintenance of neonatal primary murine intestinal organoids in vitro; however, the achievement of contractile behavior within cultured organoids has thus far been infrequent and unpredictable. Here we combine finite element simulation of oxygen transport and quantitative comparative analysis of cellular microenvironments to elucidate the critical variables that promote reproducible intestinal organoid contraction. Experimentally, oxygen distribution was manipulated by adjusting the ambient oxygen concentration along with the use of semi-permeable membranes to enhance transport. The culture microenvironment was further tailored through variation of collagen type-I matrix density, addition of exogenous R-spondin1, and specification of culture geometry. “Air–liquid interface” cultures resulted in significantly higher numbers of contractile cultures relative to traditional submerged cultures. These interface cultures were confirmed to have enhanced and more symmetric oxygen transport relative to traditional submerged cultures. While oxygen availability was found to impact in vitro contraction rate and the orientation of contractile movement, it was not a key factor in enabling contractility. For all conditions tested, reproducible contractile behavior only occurred within a consistent and narrow range of collagen type-I matrix densities with porosities of approximately 20% and storage moduli near 30 Pa. This suggests that matrix density acts as a “permissive switch” that enables contractions to occur. Similarly, contractions were only observed in cultures with diameters less than 15.5 mm that had relatively large interfacial surface area between the compliant matrix and the rigid culture dish. Taken together, these data suggest that spatial geometry and mechanics of the microenvironment, which includes both the encapsulating matrix as well as the surrounding culture device, may be key determinants of intestinal organoid functionality. As peristaltic contractility is a crucial requirement for normal digestive tract function, this achievement of reproducible organoid contraction marks a pivotal advancement towards engineering physiologically functional replacement tissue constructs.
Co-reporter:Huiyuan Wang, Lei Cai, Alexandra Paul, Annika Enejder, and Sarah C. Heilshorn
Biomacromolecules 2014 Volume 15(Issue 9) pp:
Publication Date(Web):August 11, 2014
DOI:10.1021/bm500969d
Hydrogels have been developed as extracellular matrix (ECM) mimics both for therapeutic applications and basic biological studies. In particular, elastin-like polypeptide (ELP) hydrogels, which can be tuned to mimic several biochemical and physical characteristics of native ECM, have been constructed to encapsulate various types of cells to create in vitro mimics of in vivo tissues. However, ELP hydrogels become opaque at body temperature because of ELP’s lower critical solution temperature behavior. This opacity obstructs light-based observation of the morphology and behavior of encapsulated cells. In order to improve the transparency of ELP hydrogels for better imaging, we have designed a hybrid ELP-polyethylene glycol (PEG) hydrogel system that rapidly cross-links with tris(hydroxymethyl) phosphine (THP) in aqueous solution via Mannich-type condensation. As expected, addition of the hydrophilic PEG component significantly improves the light transmittance. Coherent anti-Stokes Raman scattering (CARS) microscopy reveals that the hybrid ELP-PEG hydrogels have smaller hydrophobic ELP aggregates at 37 °C. Importantly, this hydrogel platform enables independent tuning of adhesion ligand density and matrix stiffness, which is desirable for studies of cell–matrix interactions. Human fibroblasts encapsulated in these hydrogels show high viability (>98%) after 7 days of culture. High-resolution confocal microscopy of encapsulated fibroblasts reveals that the cells adopt a more spread morphology in response to higher RGD ligand concentrations and softer gel mechanics.
Co-reporter:Alia P. Schoen, Kelly N. L. Huggins and Sarah C. Heilshorn
Journal of Materials Chemistry A 2013 vol. 1(Issue 48) pp:6662-6669
Publication Date(Web):04 Nov 2013
DOI:10.1039/C3TB21145B
The use of biomolecules to direct nanomaterial synthesis has been an area of growing interest due to the complexity of structures that can be achieved in naturally occurring systems. We previously reported the functionalization of self-assembled clathrin protein cages to enable synthesis of nanoparticles from a range of inorganic materials. Here, we investigate the ability of this engineered biomolecule complex to act as a tunable nanoreactor for the formation of different arrangements of gold nanoparticles in three dimensions. We find that self-assembled clathrin cages functionalized with engineered bi-functional peptides induce formation of gold nanoparticles to generate solutions of either dispersed or clustered gold nanoparticles on demand. The 3D arrangement of nanoparticles is dependent on the concentration of the engineered peptide, which fulfills multiple roles in the synthesis process including stabilization of the nanoparticle surface and localization of the nanoparticles within the self-assembled clathrin cage. We propose and evaluate a mechanism that allows us to predict the peptide concentration at which the nanoreactor behavior switches. This work provides insight into peptide-based surfactants and the potential for incorporating them into strategies for tuning biological mineralization processes in mild solution conditions to generate complex structures.
Co-reporter:Patrick L. Benitez;Jeffrey A. Sweet;Helen Fink;Krishna P. Chennazhi;Shantikumar V. Nair;Annika Enejder
Advanced Healthcare Materials 2013 Volume 2( Issue 1) pp:114-118
Publication Date(Web):
DOI:10.1002/adhm.201200115
Co-reporter:Andreina Parisi-Amon;Widya Mulyasasmita;Cindy Chung
Advanced Healthcare Materials 2013 Volume 2( Issue 3) pp:428-432
Publication Date(Web):
DOI:10.1002/adhm.201200293
Co-reporter:Kyle J. Lampe, Alexander L. Antaris, Sarah C. Heilshorn
Acta Biomaterialia 2013 Volume 9(Issue 3) pp:5590-5599
Publication Date(Web):March 2013
DOI:10.1016/j.actbio.2012.10.033
Abstract
The design of bioactive materials allows tailored studies probing cell–biomaterial interactions, however, relatively few studies have examined the effects of ligand density and material stiffness on neurite growth in three-dimensions. Elastin-like proteins (ELPs) have been designed with modular bioactive and structural regions to enable the systematic characterization of design parameters within three-dimensional (3-D) materials. To promote neurite out-growth and better understand the effects of common biomaterial design parameters on neuronal cultures we here focused on the cell-adhesive ligand density and hydrogel stiffness as design variables for ELP hydrogels. With the inherent design freedom of engineered proteins these 3-D ELP hydrogels enabled decoupled investigations into the effects of biomechanics and biochemistry on neurite out-growth from dorsal root ganglia. Increasing the cell-adhesive RGD ligand density from 0 to 1.9 × 107 ligands μm−3 led to a significant increase in the rate, length, and density of neurite out-growth, as quantified by a high throughput algorithm developed for dense neurite analysis. An approximately two-fold improvement in total neurite out-growth was observed in materials with the higher ligand density at all time points up to 7 days. ELP hydrogels with initial elastic moduli of 0.5, 1.5, or 2.1 kPa and identical RGD ligand densities revealed that the most compliant materials led to the greatest out-growth, with some neurites extending over 1800 μm by day 7. Given the ability of ELP hydrogels to efficiently promote neurite out-growth within defined and tunable 3-D microenvironments these materials may be useful in developing therapeutic nerve guides and the further study of basic neuron–biomaterial interactions.
Co-reporter:Cindy Chung, Beth L. Pruitt and Sarah C. Heilshorn
Biomaterials Science 2013 vol. 1(Issue 10) pp:1082-1090
Publication Date(Web):15 Jul 2013
DOI:10.1039/C3BM60139K
Cellular therapies have great potential to provide alternative treatment options for those suffering from heart disease. In order to optimize cell delivery for therapeutic efficacy, a greater understanding of parameters that impact stem cell differentiation, survival, growth, and development are needed. In this study, we examine the role of hydrogel crosslink density on spontaneous cardiomyocyte (CM) differentiation of murine embryoid bodies (EBs). CM differentiation was accelerated in hydrogels of low crosslink density, where 100% of the hydrogels were positive for CM differentiation compared to only 53% in the high crosslink density group after 8 days of culture. DNA microarray data suggests that enhanced CM differentiation in the low crosslink density hydrogels was not tissue specific but rather a result of favoured EB development and cell proliferation. Additionally, enhanced EB growth and differentiation in low crosslink density hydrogels was independent of RGD ligand density and not a consequence of enhanced diffusion. We also demonstrate that matrix metalloproteinase activity is required for spontaneous CM differentiation in 3D hydrogels. Low hydrogel crosslink density regulates spontaneous EB differentiation by promoting EB growth and development. Elucidating the effects of microenvironmental cues on cell differentiation can aid in the optimization of stem cell-based therapies for tissue regeneration.
Co-reporter:Alia P. Schoen, Nicholas Cordella, Shafigh Mehraeen, Manickam Adhimoolam Arunagirinathan, Andrew J. Spakowitz and Sarah C. Heilshorn
Soft Matter 2013 vol. 9(Issue 38) pp:9137-9145
Publication Date(Web):30 May 2013
DOI:10.1039/C3SM50830G
Clathrin is a naturally evolved protein that robustly assembles and disassembles into nanoscale spherical cages. This ability to reorganize in a highly dynamic fashion makes clathrin an attractive model system to study the kinetic and thermodynamic principles of biomolecular self-assembly. Through a combination of experimental and computational approaches, we demonstrate that competition between weak non-specific and specific reversible interactions can dictate the initial pathway of the assembly process, yet the final assembled structures are not sensitive to this competition. We conclude that the relative strengths of non-specific and specific interactions control clathrin assembly at short time scales resulting in either disordered protein aggregates or regularly structured assemblies. However with sufficient time for remodeling, the final assembled structure is robustly formed due to geometric constraints arising from specific molecular recognition events. These data provide insight into naturally evolved biological assembly processes and guidance for the design of engineered systems to achieve robust assembly.
Co-reporter:Amir Shamloo, Milan Manchandia, Meghaan Ferreira, Maheswaran Mani, Christopher Nguyen, Thomas Jahn, Kenneth Weinberg and Sarah Heilshorn
Integrative Biology 2013 vol. 5(Issue 8) pp:1076-1085
Publication Date(Web):17 Jun 2013
DOI:10.1039/C3IB40025E
Besides its cooperating effects on stem cell proliferation and survival, Kit ligand (KL) is a potent chemotactic protein. While transwell assays permit studies of the frequency of migrating cells, the lack of direct visualization precludes dynamic chemotaxis studies. In response, we utilize microfluidic chambers that enable direct observation of murine bone marrow-derived mast cells (BMMC) within stable KL gradients. Using this system, individual Kit+ BMMC were quantitatively analyzed for migration speed and directionality during KL-induced chemotaxis. Our results indicated a minimum activating threshold of ∼3 ng ml−1 for chemoattraction. Analysis of cells at KL concentrations below 3 ng ml−1 revealed a paradoxical chemorepulsion, which has not been described previously. Unlike chemoattraction, which occurred continuously after an initial time lag, chemorepulsion occurred only during the first 90 minutes of observation. Both chemoattraction and chemorepulsion required the action of G-protein coupled receptors (GPCR), as treatment with pertussis toxin abrogated directed migration. These results differ from previous studies of GPCR-mediated chemotaxis, where chemorepulsion occurred at high ligand concentrations. These data indicate that Kit-mediated chemotaxis is more complex than previously understood, with the involvement of GPCRs in addition to the Kit receptor tyrosine kinase and the presence of both chemoattractive and chemorepellent phases.
Co-reporter:Rebecca L. DiMarco
Advanced Materials 2012 Volume 24( Issue 29) pp:3923-3940
Publication Date(Web):
DOI:10.1002/adma.201200051
Abstract
The diversity of potential applications for protein-engineered materials has undergone profound recent expansion through a rapid increase in the library of domains that have been utilized in these materials. Historically, protein-engineered biomaterials have been generated from a handful of peptides that were selected and exploited for their naturally evolved functionalities. In recent years, the scope of the field has drastically expanded to include peptide domains that were designed through computational modeling, identified through high-throughput screening, or repurposed from wild type domains to perform functions distinct from their primary native applications. The strategy of exploiting a diverse library of peptide domains to design modular block copolymers enables the synthesis of multifunctional protein-engineered materials with a range of customizable properties and activities. As the diversity of peptide domains utilized in modular protein engineering continues to expand, a tremendous and ever-growing combinatorial expanse of material functionalities will result.
Co-reporter:Jordan Raphel, Andreina Parisi-Amon and Sarah C. Heilshorn
Journal of Materials Chemistry A 2012 vol. 22(Issue 37) pp:19429-19437
Publication Date(Web):26 Apr 2012
DOI:10.1039/C2JM31768K
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 nanometre to millimetre 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.
Co-reporter:Debanti Sengupta;Penney M. Gilbert;Kyle J. Johnson;Helen M. Blau
Advanced Healthcare Materials 2012 Volume 1( Issue 6) pp:785-789
Publication Date(Web):
DOI:10.1002/adhm.201200195
Co-reporter:Cindy Chung, Erica Anderson, Renee Reijo Pera, Beth L. Pruitt and Sarah C. Heilshorn
Soft Matter 2012 vol. 8(Issue 39) pp:10141-10148
Publication Date(Web):21 Aug 2012
DOI:10.1039/C2SM26082D
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 for up to 6 days. However, once spontaneous contraction was restored, the rate of contraction was similar within all materials (71 ± 8 beats per 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.
Co-reporter:Cindy Chung, Kyle J. Lampe, and Sarah C. Heilshorn
Biomacromolecules 2012 Volume 13(Issue 12) pp:
Publication Date(Web):November 14, 2012
DOI:10.1021/bm3015279
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 cross-linking must be mild and cell compatible. While many chemical cross-linkers are commercially available for hydrogel formation, only a subset are cytocompatible; therefore, the identification of new and reliable cytocompatible cross-linkers 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 cross-linker for 3D cell encapsulation in protein-based hydrogels. We characterize the THPC-amine reaction by demonstrating THPC's 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 with two cell types, embryonic stem cells and neuronal cells, where cells exhibited the ability to differentiate and grow in elastin-like protein hydrogels. The primary goal of this communication is to report the identification and utility of tetrakis(hydroxymethyl) phosphonium chloride (THPC) as an inexpensive but widely applicable cross-linker for protein-based materials.
Co-reporter:J. Tanner Nevill, Alexander Mo, Branden J. Cord, Theo D. Palmer, Mu-ming Poo, Luke P. Lee and Sarah C. Heilshorn
Soft Matter 2011 vol. 7(Issue 2) pp:343-347
Publication Date(Web):25 Oct 2010
DOI:10.1039/C0SM00869A
The ability to coat surfaces with pre-determined patterns of biomolecules by soft lithography has found use in areas ranging from fundamental biology to translational medicine, such as tissue engineering and diagnostics. However, existing surface patterning techniques (e.g., microcontact printing and traditional lithography) are unable to pattern several biomolecules in a single step. Here we introduce a simple method to simultaneously pattern multiple biomolecules in complex two-dimensional configurations onto substrates with better than 2 µm resolution. This protocol, termed vacuum soft lithography, utilized below ambient pressures temporarily stored within a removable microfluidic template to expose specific regions of a substrate to multiple biochemical solutions. We demonstrate the utility of this vacuum soft lithography technique by fabricating a multi-component array that directs the adhesion, polarization, and neurite guidance of primary hippocampal neurons.
Co-reporter:Widya Mulyasasmita, Ji Seok Lee, and Sarah C. Heilshorn
Biomacromolecules 2011 Volume 12(Issue 10) pp:
Publication Date(Web):August 23, 2011
DOI:10.1021/bm200959e
Predictable tuning of bulk mechanics from the molecular level remains elusive in many physical hydrogel systems because of the reliance on nonspecific and nonstoichiometric 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 a simple polymer physics model, we manipulate the polypeptide binding interactions and demonstrate the direct ability to predict the resulting effects on network cross-linking density, sol–gel phase behavior, and gel mechanics.
Co-reporter:Amir Shamloo and Sarah C. Heilshorn
Lab on a Chip 2010 vol. 10(Issue 22) pp:3061-3068
Publication Date(Web):01 Sep 2010
DOI:10.1039/C005069E
Endothelial cell (EC) sprouting morphogenesis is a critical step during angiogenesis, the formation of new blood vessels from existing conduits. Here, three-dimensional sprouting morphogenesis was examined using in vitro microfluidic devices that enabled the separate and simultaneous tuning of biomechanical and soluble biochemical stimuli. Quantitative analysis of endothelial sprout formation demonstrated that the ability of vascular endothelial growth factor (VEGF) to regulate stable sprout formation was mediated by the density of the surrounding collagen/fibronectin matrix. The coordinated migration and proliferation of multiple ECs to form stable sprouts were enhanced at intermediate matrix densities (1.2–1.9 mg ml−1), while lower densities resulted in uncoordinated migration (0.3–0.7 mg ml−1) and higher densities resulted in broad cell clusters that did not elongate (2.7 mg ml−1). Within the permissive range of matrix biomechanics, higher density matrices resulted in shorter, thicker, and slower-growing sprouts. The sprouts in higher density matrices also were more likely to polarize towards higher VEGF concentrations, included more cells per cross-sectional area, and demonstrated more stable lumen formation compared to sprouts in lower density matrices. These results quantitatively demonstrate that matrix density mediates VEGF-induced sprout polarization and lumen formation, potentially by regulating the balance between EC migration rate and proliferation rate.
Co-reporter:Karin S. Straley
Advanced Materials 2009 Volume 21( Issue 41) pp:4148-4152
Publication Date(Web):
DOI:10.1002/adma.200901865
Co-reporter:Karin S. Straley
Advanced Materials 2009 Volume 21( Issue 41) pp:
Publication Date(Web):
DOI:10.1002/adma.200990152
Co-reporter:Karin S. Straley and Sarah C. Heilshorn
Soft Matter 2009 vol. 5(Issue 1) pp:114-124
Publication Date(Web):23 Oct 2008
DOI:10.1039/B808504H
A key attribute missing from many current biomaterials is the ability to independently tune multiple biomaterial properties without simultaneously affecting other material parameters. Because cells are well known to respond to changes in the initial elastic modulus, degradation rate, and cell adhesivity of a biomaterial, it is critical to develop synthetic design strategies that allow decoupled tailoring of each individual parameter in order to systematically optimize cell-scaffold interactions. We present the development of a biomimetic scaffold composed of chemically crosslinked, elastin-like proteins designed to support neural regeneration through a combination of cell adhesion and cell-induced degradation and remodeling. The design of these engineered proteins includes cell adhesion sequences to enable neuronal attachment as well as sequences sensitive to cleavage by urokinase plasminogen activator (uPA), a protease locally secreted from the tips of growing neurites, to enable highly localized and tunable degradation properties. These engineered proteins are produced using recombinant techniques and chemically crosslinked into highly swollen hydrogels with controllable mechanical properties. Through a modest 3% change in the chemical identity of three otherwise identical engineered proteins, we can modify the uPA substrate specificity resulting in tunable changes in protease degradation half-life over two orders of magnitude. Under high uPA exposure, the designed scaffolds exhibit systematic variation of scaffold lifetime, from being fully degraded within a single day to showing no noticeable degradation within a full week. In vitro studies using the model PC-12 neuronal-like cell line show that the crosslinked proteins support tunable cell adhesion and neuronal differentiation. Increasing the density of RGD peptides present in the protein substrates leads to increased cell adhesion and more extensive neurite outgrowth. These engineered proteins offer the ability to independently tailor the mechanics, degradation properties, and cell adhesivity of scaffolds for the study of central nervous system regeneration.
Co-reporter:Ji Seok Lee;Widya Mulyasasmita;Cheryl T. S. Wong Po Foo;Andreina Parisi-Amon
PNAS 2009 Volume 106 (Issue 52 ) pp:22067-22072
Publication Date(Web):2009-12-29
DOI:10.1073/pnas.0904851106
Current protocols to encapsulate cells within physical hydrogels require substantial changes in environmental conditions (pH,
temperature, or ionic strength) to initiate gelation. These conditions can be detrimental to cells and are often difficult
to reproduce, therefore complicating their use in clinical settings. We report the development of a two-component, molecular-recognition
gelation strategy that enables cell encapsulation without environmental triggers. Instead, the two components, which contain
multiple repeats of WW and proline-rich peptide domains, undergo a sol–gel phase transition upon simple mixing and hetero-assembly
of the peptide domains. We term these materials mixing-induced, two-component hydrogels. Our results demonstrate use of the
WW and proline-rich domains in protein-engineered materials and expand the library of peptides successfully designed into
engineered proteins. Because both of these association domains are normally found intracellularly, their molecular recognition
is not disrupted by the presence of additional biomolecules in the extracellular milieu, thereby enabling reproducible encapsulation
of multiple cell types, including PC-12 neuronal-like cells, human umbilical vein endothelial cells, and murine adult neural
stem cells. Precise variations in the molecular-level design of the two components including (i) the frequency of repeated association domains per chain and (ii) the association energy between domains enable tailoring of the hydrogel viscoelasticity to achieve plateau shear moduli
ranging from ≈9 to 50 Pa. Because of the transient physical crosslinks that form between association domains, these hydrogels
are shear-thinning, injectable, and self-healing. Neural stem cells encapsulated in the hydrogels form stable three-dimensional
cultures that continue to self-renew, differentiate, and sprout extended neurites.
Co-reporter:Sheng Wang;Cheryl Wong Po Foo;Ajithkumar Warrier;Mu-ming Poo
Biomedical Microdevices 2009 Volume 11( Issue 5) pp:
Publication Date(Web):2009 October
DOI:10.1007/s10544-009-9329-1
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.
Co-reporter:Amir Shamloo, Ning Ma, Mu-ming Poo, Lydia L. Sohn and Sarah C. Heilshorn
Lab on a Chip 2008 vol. 8(Issue 8) pp:1292-1299
Publication Date(Web):30 May 2008
DOI:10.1039/B719788H
The directed migration of endothelial cells is an early and critical step in angiogenesis, or new blood vessel formation. In this study, the polarization and chemotaxis of human umbilical vein endothelial cells (HUVEC) in response to quantified gradients of vascular endothelial growth factor (VEGF) were examined. To accomplish this, a microfluidic device was designed and fabricated to generate stable concentration gradients of biomolecules in a cell culture chamber while minimizing the fluid shear stress experienced by the cells. Finite element simulation of the device geometry produced excellent agreement with the observed VEGF concentration distribution, which was found to be stable across multiple hours. This device is expected to have wide applicability in the study of shear-sensitive cells such as HUVEC and non-adherent cell types as well as in the study of migration through three-dimensional matrices. HUVEC were observed to chemotax towards higher VEGF concentrations across the entire range of concentrations studied (18–32 ng mL−1) when the concentration gradient was 14 ng mL−1 mm−1. In contrast, shallow gradients (2 ng mL−1 mm−1) across the same concentration range were unable to induce HUVEC chemotaxis. Furthermore, while all HUVEC exposed to elevated VEGF levels (both in steep and shallow gradients) displayed an increased number of filopodia, only chemotaxing HUVEC displayed an asymmetric distribution of filopodia, with enhanced numbers of protrusions present along the leading edge. These results suggest a two-part requirement to induce VEGF chemotaxis: the VEGF absolute concentration enhances the total number of filopodia extended while the VEGF gradient steepness induces filopodia localization, cell polarization, and subsequent directed migration.
Co-reporter:Danqing Zhu, Huiyuan Wang, Pavin Trinh, Sarah C. Heilshorn, Fan Yang
Biomaterials (May 2017) Volume 127() pp:132-140
Publication Date(Web):May 2017
DOI:10.1016/j.biomaterials.2017.02.010
Co-reporter:Jordan Raphel, Johan Karlsson, Silvia Galli, Ann Wennerberg, Christopher Lindsay, Matthew G. Haugh, Jukka Pajarinen, Stuart B. Goodman, Ryo Jimbo, Martin Andersson, Sarah C. Heilshorn
Biomaterials (March 2016) Volume 83() pp:269-282
Publication Date(Web):March 2016
DOI:10.1016/j.biomaterials.2015.12.030
Here we present the design of an engineered, elastin-like protein (ELP) that is chemically modified to enable stable coatings on the surfaces of titanium-based dental and orthopaedic implants by novel photocrosslinking and solution processing steps. The ELP includes an extended RGD sequence to confer bio-signaling and an elastin-like sequence for mechanical stability. ELP thin films were fabricated on cp-Ti and Ti6Al4V surfaces using scalable spin and dip coating processes with photoactive covalent crosslinking through a carbene insertion mechanism. The coatings withstood procedures mimicking dental screw and hip replacement stem implantations, a key metric for clinical translation. They promoted rapid adhesion of MG63 osteoblast-like cells, with over 80% adhesion after 24 h, compared to 38% adhesion on uncoated Ti6Al4V. MG63 cells produced significantly more mineralization on ELP coatings compared to uncoated Ti6Al4V. Human bone marrow mesenchymal stem cells (hMSCs) had an earlier increase in alkaline phosphatase activity, indicating more rapid osteogenic differentiation and mineral deposition on adhesive ELP coatings. Rat tibia and femur in vivo studies demonstrated that cell-adhesive ELP-coated implants increased bone-implant contact area and interfacial strength after one week. These results suggest that ELP coatings withstand surgical implantation and promote rapid osseointegration, enabling earlier implant loading and potentially preventing micromotion that leads to aseptic loosening and premature implant failure.
Co-reporter:Jordan Raphel, Johan Karlsson, Silvia Galli, Ann Wennerberg, Christopher Lindsay, Matthew G. Haugh, Jukka Pajarinen, Stuart B. Goodman, Ryo Jimbo, Martin Andersson, Sarah C. Heilshorn
Biomaterials (March 2016) Volume 83() pp:
Publication Date(Web):March 2016
DOI:10.1016/j.biomaterials.2015.12.030
Here we present the design of an engineered, elastin-like protein (ELP) that is chemically modified to enable stable coatings on the surfaces of titanium-based dental and orthopaedic implants by novel photocrosslinking and solution processing steps. The ELP includes an extended RGD sequence to confer bio-signaling and an elastin-like sequence for mechanical stability. ELP thin films were fabricated on cp-Ti and Ti6Al4V surfaces using scalable spin and dip coating processes with photoactive covalent crosslinking through a carbene insertion mechanism. The coatings withstood procedures mimicking dental screw and hip replacement stem implantations, a key metric for clinical translation. They promoted rapid adhesion of MG63 osteoblast-like cells, with over 80% adhesion after 24 h, compared to 38% adhesion on uncoated Ti6Al4V. MG63 cells produced significantly more mineralization on ELP coatings compared to uncoated Ti6Al4V. Human bone marrow mesenchymal stem cells (hMSCs) had an earlier increase in alkaline phosphatase activity, indicating more rapid osteogenic differentiation and mineral deposition on adhesive ELP coatings. Rat tibia and femur in vivo studies demonstrated that cell-adhesive ELP-coated implants increased bone-implant contact area and interfacial strength after one week. These results suggest that ELP coatings withstand surgical implantation and promote rapid osseointegration, enabling earlier implant loading and potentially preventing micromotion that leads to aseptic loosening and premature implant failure.
Co-reporter:Shamik Mascharak, Patrick L. Benitez, Amy C. Proctor, Christopher M. Madl, Kenneth H. Hu, Ruby E. Dewi, Manish J. Butte, Sarah C. Heilshorn
Biomaterials (January 2017) Volume 115() pp:155-166
Publication Date(Web):January 2017
DOI:10.1016/j.biomaterials.2016.11.019
Native vascular extracellular matrices (vECM) consist of elastic fibers that impart varied topographical properties, yet most in vitro models designed to study the effects of topography on cell behavior are not representative of native architecture. Here, we engineer an electrospun elastin-like protein (ELP) system with independently tunable, vECM-mimetic topography and demonstrate that increasing topographical variation causes loss of endothelial cell-cell junction organization. This loss of VE-cadherin signaling and increased cytoskeletal contractility on more topographically varied ELP substrates in turn promote YAP activation and nuclear translocation, resulting in significantly increased endothelial cell migration and proliferation. Our findings identify YAP as a required signaling factor through which fibrous substrate topography influences cell behavior and highlights topography as a key design parameter for engineered biomaterials.
Co-reporter:Shamik Mascharak, Patrick L. Benitez, Amy C. Proctor, Christopher M. Madl, Kenneth H. Hu, Ruby E. Dewi, Manish J. Butte, Sarah C. Heilshorn
Biomaterials (January 2017) Volume 115() pp:155-166
Publication Date(Web):January 2017
DOI:10.1016/j.biomaterials.2016.11.019
Co-reporter:Abbygail A Foster, Laura M Marquardt, Sarah C Heilshorn
Current Opinion in Chemical Engineering (February 2017) Volume 15() pp:15-23
Publication Date(Web):1 February 2017
DOI:10.1016/j.coche.2016.11.003
•Stem cell transplantation by local injection has seen limited clinical success.•Different transplantation stages present different mechanical challenges.•Hydrogels with tunable mechanics can overcome mechanical challenges at each stage.Stem cell delivery by local injection has tremendous potential as a regenerative therapy but has seen limited clinical success. Several mechanical challenges hinder therapeutic efficacy throughout all stages of cell transplantation, including mechanical forces during injection and loss of mechanical support post-injection. Recent studies have begun exploring the use of biomaterials, in particular hydrogels, to enhance stem cell transplantation by addressing the often-conflicting mechanical requirements associated with each stage of the transplantation process. This review explores recent biomaterial approaches to improve the therapeutic efficacy of stem cells delivered through local injection, with a focus on strategies that specifically address the mechanical challenges that result in cell death and/or limit therapeutic function throughout the stages of transplantation.
Co-reporter:Kyle J. Lampe, Sarah C. Heilshorn
Neuroscience Letters (25 June 2012) Volume 519(Issue 2) pp:138-146
Publication Date(Web):25 June 2012
DOI:10.1016/j.neulet.2012.01.042
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.Highlights► Designing mimics of the neural stem cell niche through biomaterial engineering. ► Molecular design of peptides and proteins that mimic the extracellular matrix. ► Polypeptide hydrogels for three-dimensional neural stem cell culture. ► Engineered biopolymers suitable for delivery of transplanted stem cells. ► Pre-clinical and clinical trials of engineered polypeptide materials within the CNS.
Co-reporter:Jordan Raphel, Andreina Parisi-Amon and Sarah C. Heilshorn
Journal of Materials Chemistry A 2012 - vol. 22(Issue 37) pp:NaN19437-19437
Publication Date(Web):2012/04/26
DOI:10.1039/C2JM31768K
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 nanometre to millimetre 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.
Co-reporter:Alia P. Schoen, Kelly N. L. Huggins and Sarah C. Heilshorn
Journal of Materials Chemistry A 2013 - vol. 1(Issue 48) pp:NaN6669-6669
Publication Date(Web):2013/11/04
DOI:10.1039/C3TB21145B
The use of biomolecules to direct nanomaterial synthesis has been an area of growing interest due to the complexity of structures that can be achieved in naturally occurring systems. We previously reported the functionalization of self-assembled clathrin protein cages to enable synthesis of nanoparticles from a range of inorganic materials. Here, we investigate the ability of this engineered biomolecule complex to act as a tunable nanoreactor for the formation of different arrangements of gold nanoparticles in three dimensions. We find that self-assembled clathrin cages functionalized with engineered bi-functional peptides induce formation of gold nanoparticles to generate solutions of either dispersed or clustered gold nanoparticles on demand. The 3D arrangement of nanoparticles is dependent on the concentration of the engineered peptide, which fulfills multiple roles in the synthesis process including stabilization of the nanoparticle surface and localization of the nanoparticles within the self-assembled clathrin cage. We propose and evaluate a mechanism that allows us to predict the peptide concentration at which the nanoreactor behavior switches. This work provides insight into peptide-based surfactants and the potential for incorporating them into strategies for tuning biological mineralization processes in mild solution conditions to generate complex structures.
Co-reporter:Cindy Chung, Beth L. Pruitt and Sarah C. Heilshorn
Biomaterials Science (2013-Present) 2013 - vol. 1(Issue 10) pp:NaN1090-1090
Publication Date(Web):2013/07/15
DOI:10.1039/C3BM60139K
Cellular therapies have great potential to provide alternative treatment options for those suffering from heart disease. In order to optimize cell delivery for therapeutic efficacy, a greater understanding of parameters that impact stem cell differentiation, survival, growth, and development are needed. In this study, we examine the role of hydrogel crosslink density on spontaneous cardiomyocyte (CM) differentiation of murine embryoid bodies (EBs). CM differentiation was accelerated in hydrogels of low crosslink density, where 100% of the hydrogels were positive for CM differentiation compared to only 53% in the high crosslink density group after 8 days of culture. DNA microarray data suggests that enhanced CM differentiation in the low crosslink density hydrogels was not tissue specific but rather a result of favoured EB development and cell proliferation. Additionally, enhanced EB growth and differentiation in low crosslink density hydrogels was independent of RGD ligand density and not a consequence of enhanced diffusion. We also demonstrate that matrix metalloproteinase activity is required for spontaneous CM differentiation in 3D hydrogels. Low hydrogel crosslink density regulates spontaneous EB differentiation by promoting EB growth and development. Elucidating the effects of microenvironmental cues on cell differentiation can aid in the optimization of stem cell-based therapies for tissue regeneration.
Co-reporter:Midori Greenwood-Goodwin, Eric S. Teasley and Sarah C. Heilshorn
Biomaterials Science (2013-Present) 2014 - vol. 2(Issue 11) pp:NaN1639-1639
Publication Date(Web):2014/08/07
DOI:10.1039/C4BM00142G
Engineered biomimetic microenvironments from hydrogels are an emerging strategy to achieve lineage-specific differentiation in vitro. In addition to recapitulating critical matrix cues found in the native three-dimensional (3D) niche, the hydrogel can also be designed to deliver soluble factors that are present within the native inductive microenvironment. We demonstrate a versatile materials approach for the dual-stage delivery of multiple soluble factors within a 3D hydrogel to induce adipogenesis. We use a mixing-induced two-component hydrogel (MITCH) embedded with alginate microgels to deliver two pro-adipogenic soluble factors, fibroblast growth factor 1 (FGF-1) and bone morphogenetic protein 4 (BMP-4) with two distinct delivery profiles. We show that dual-stage delivery of FGF-1 and BMP-4 to human adipose-derived stromal cells (hADSCs) significantly increases lipid accumulation compared with the simultaneous delivery of both growth factors together. Furthermore, dual-stage growth factor delivery within a 3D hydrogel resulted in substantially more lipid accumulation compared to identical delivery profiles in 2D cultures. Gene expression analysis shows upregulation of key adipogenic markers indicative of brown-like adipocytes. These data suggest that dual-stage release of FGF-1 and BMP-4 within 3D microenvironments can promote the in vitro development of mature adipocytes.
Co-reporter:Lei Cai, Cong B. Dinh and Sarah C. Heilshorn
Biomaterials Science (2013-Present) 2014 - vol. 2(Issue 5) pp:NaN765-765
Publication Date(Web):2014/02/04
DOI:10.1039/C3BM60293A
Immobilization of growth factors to polymeric matrices has been a common strategy in the design of tissue engineering scaffolds to promote tissue regeneration, which requires complex cell signaling events with the surrounding matrix. However, the use of large protein growth factors in polymeric scaffolds is often plagued by immunogenicity, short in vivo half-lives, and reduced bioactivity. To address these concerns, we developed a single-step, cell-compatible strategy to tether small, growth-factor-mimetic peptides into a protein-engineered hydrogel with tunable biomaterial properties. Specifically, we covalently immobilized the QK peptide, an angiogenic peptide mimicking the receptor-binding region of vascular endothelial growth factor (VEGF), within tunable elastin-like polypeptide (ELP) hydrogels that include a cell-adhesive RGD sequence. Using a cell-compatible, amine-reactive crosslinker, we conducted a one-pot synthesis to simultaneously encapsulate cells while precisely controlling the QK grafting density (10 nM–100 μM) in the ELP hydrogels without altering other material properties. Fluorescence analysis of fluor-labeled QK peptides demonstrated that the conjugation efficiency to ELP hydrogels was >75% and that covalent immobilization effectively eliminates all QK diffusion. Compared with pristine ELP hydrogels, human umbilical vein endothelial cell (HUVEC) proliferation was significantly enhanced on ELP hydrogels immobilized with 10 nM or 1 μM QK. Moreover, upon encapsulation within tethered QK-ELP hydrogels, HUVEC spheroids maintained near 100% viability and demonstrated significantly more three-dimensional outgrowth compared to those supplemented with soluble QK peptide at the same concentration. These results encourage the further development of protein-engineered scaffolds decorated with growth-factor-mimetic peptides to provide long-term biological signals using this versatile, single-step synthesis.
Co-reporter:Rebecca L. DiMarco, Ruby E. Dewi, Gabriela Bernal, Calvin Kuo and Sarah C. Heilshorn
Biomaterials Science (2013-Present) 2015 - vol. 3(Issue 10) pp:NaN1385-1385
Publication Date(Web):2015/07/16
DOI:10.1039/C5BM00108K
Though in vitro culture of primary intestinal organoids has gained significant momentum in recent years, little has been done to investigate the impact of microenvironmental cues provided by the encapsulating matrix on the growth and development of these fragile cultures. In this work, the impact of various in vitro culture parameters on primary adult murine organoid formation and growth are analyzed with a focus on matrix properties and geometric culture configuration. The air–liquid interface culture configuration was found to result in enhanced organoid formation relative to a traditional submerged configuration. Additionally, through use of a recombinantly engineered extracellular matrix (eECM), the effects of biochemical and biomechanical cues were independently studied. Decreasing mechanical stiffness and increasing cell adhesivity were found to increase organoid yield. Tuning of eECM properties was used to obtain organoid formation efficiency values identical to those observed in naturally harvested collagen I matrices but within a stiffer construct with improved ease of physical manipulation. Increased ability to remodel the surrounding matrix through mechanical or enzymatic means was also shown to enhance organoid formation. As the engineering and tunability of recombinant matrices is essentially limitless, continued property optimization may result in further improved matrix performance and may help to identify additional microenvironmental cues that directly impact organoid formation, development, differentiation, and functional behavior. Continued culture of primary organoids in recombinant matrices could therefore prove to be largely advantageous in the field of intestinal tissue engineering for applications in regenerative medicine and in vitro tissue mimics.
