Doctor of Philosophy, University of Tennessee (2012)
Bachelor of Engineering, Zhejiang University (2008)
Sarah Heilshorn, Postdoctoral Faculty Sponsor
Avidity-controlled hydrogels for injectable co-delivery of induced pluripotent stem cell-derived endothelial cells and growth factors.
Journal of controlled release
2014; 191: 71-81
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 20kDa and 40kDa 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 20kDa PEG variants exhibited slower release of encapsulated vascular endothelial growth factor (VEGF), due to a decrease in hydrogel mesh size relative to the 40kDa 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.
View details for DOI 10.1016/j.jconrel.2014.05.015
View details for PubMedID 24848744
- Hybrid Elastin-like Polypeptide-Polyethylene Glycol (ELP-PEG) Hydrogels with Improved Transparency and Independent Control of Matrix Mechanics and Cell Ligand Density BIOMACROMOLECULES 2014; 15 (9): 3421-3428
Avidity-controlled delivery of angiogenic peptides from injectable molecular-recognition hydrogels.
Tissue engineering. Part A
2014; 20 (15-16): 2102-2114
Peptide mimics of growth factors represent an emerging class of therapeutic drugs due to high biological specificity and relative ease of synthesis. However, maintaining efficacious therapeutic dosage at the therapy site has proven challenging owing to poor intestinal permeability and short circulating half-lives in the blood stream. In this work, we present the affinity immobilization and controlled release of QK, a vascular endothelial growth factor (VEGF) mimetic peptide, from an injectable mixing-induced two-component hydrogel (MITCH). The MITCH system is crosslinked by reversible interactions between WW domains and complementary proline-rich peptide modules. Fusion of the QK peptide to either one or two units of the proline-rich sequence creates bifunctional peptide conjugates capable of specific binding to MITCH while preserving their angiogenic bioactivity. Presenting two repeats of the proline-rich sequence increases the binding enthalpy 2.5 times due to avidity effects. Mixing of the drug conjugates with MITCH components results in drug encapsulation and extended release at rates consistent with the affinity immobilization strength. Human umbilical vein endothelial cells (HUVECs) treated with the soluble drug conjugates exhibit morphogenetic events of VEGF receptor 2 signal transduction followed by cell migration and organization into networks characteristic of early angiogenesis. In a three-dimensional model where HUVECs were cultured as spheroids in a matrix of collagen and fibronectin, injection of drug-releasing MITCH resulted in significantly more cell outgrowth than drugs injected in saline. This ability to sustain local drug availability is ideal for therapeutic angiogenesis applications, where spatiotemporal control over drug distribution is a key requirement for clinical success.
View details for DOI 10.1089/ten.tea.2013.0357
View details for PubMedID 24490588
Designing ECM-mimetic materials using protein engineering
2014; 10 (4): 1751-1760
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 20years, 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.
View details for DOI 10.1016/j.actbio.2013.12.028
View details for Web of Science ID 000334137700025
- One-pot synthesis of elastin-like polypeptide hydrogels with grafted VEGF-mimetic peptides BIOMATERIALS SCIENCE 2014; 2 (5): 757-765
Photocured Biodegradable Polymer Substrates of Varying Stiffness and Microgroove Dimensions for Promoting Nerve Cell Guidance and Differentiation
2012; 28 (34): 12557-12568
Photocross-linkable and biodegradable polymers have great promise in fabricating nerve conduits for guiding axonal growth in peripheral nerve regeneration. Here, we photocross-linked two poly(ε-caprolactone) triacrylates (PCLTAs) with number-average molecular weights of ~7000 and ~10,000 g mol(-1) into substrates with parallel microgrooves. Cross-linked PCLTA7k was amorphous and soft, while cross-linked PCLTA10k was semicrystalline with a stiffer surface. We employed different dimensions of interests for the parallel microgrooves, that is, groove widths of 5, 15, 45, and 90 μm and groove depths of 0.4, 1, 5, and 12 μm. The behaviors of rat Schwann cell precursor line (SpL201) cells with the glial nature and pheochromocytoma (PC12) cells with the neuronal nature were studied on these microgrooved substrates, showing distinct preference to the substrates with different mechanical properties. We found different threshold sensitivities of the two nerve cell types to topographical features when their cytoskeleton and nuclei were altered by varying the groove depth and width. Almost all of the cells were aligned in the narrowest and deepest microgrooves or around the edge of microgrooves. Oriented SpL201 cell movement had a higher motility as compared to unaligned ones. After forskolin treatment, SpL201 cells demonstrated significantly upregulated S-100 and O4 on stiffer substrates or narrower microgrooves, suggesting more differentiation toward early Schwann cells (SCs). PC12 neurites were oriented with enhanced extension in narrower microgrooves. The present results not only improve our fundamental understanding on nerve cell-substrate interactions, but also offer useful conduit materials and appropriate feature dimensions to foster guidance for axonal growth in peripheral nerve regeneration.
View details for DOI 10.1021/la302868q
View details for Web of Science ID 000307988700018
View details for PubMedID 22857011
- Injectable and Biodegradable Nanohybrid Polymers with Simultaneously Enhanced Stiffness and Toughness for Bone Repair ADVANCED FUNCTIONAL MATERIALS 2012; 22 (15): 3181-3190
Optimal Poly(L-lysine) Grafting Density in Hydrogels for Promoting Neural Progenitor Cell Functions
2012; 13 (5): 1663-1674
Recently, we have developed a photopolymerizable poly(L-lysine) (PLL) that can be covalently incorporated into poly(ethylene glycol) diacrylate (PEGDA) hydrogels to improve their bioactivity by providing positive charges. To explore the potential of these PLL-grafted PEGDA hydrogels as a cell delivery vehicle and luminal filler in nerve guidance conduits for peripheral and central nerve regeneration, we varied the number of pendent PLL chains in the hydrogels by photo-cross-linking PEGDA with weight compositions of PLL (φ(PLL)) of 0, 1, 2, 3, and 5%. We further investigated the effect of PLL grafting density on E14 mouse neural progenitor cell (NPC) behavior including cell viability, attachment, proliferation, differentiation, and gene expression. The amount of actually grafted PLL and charge densities were characterized, showing a proportional increase with the feed composition φ(PLL). NPC viability in 3D hydrogels was significantly improved in a PLL grafting density-dependent manner at days 7 and 14 postencapsulation. Similarly, NPC attachment and proliferation were promoted on the PLL-grafted hydrogels with increasing φ(PLL) up to 2%. More intriguingly, NPC lineage commitment was dramatically altered by the amount of grafted PLL chains in the hydrogels. NPC differentiation demonstrated a parabolic or nonmonotonic dependence on φ(PLL), resulting in cells mostly differentiated toward mature neurons with extensive neurite formation and astrocytes rather than oligodendrocytes on the PLL-grafted hydrogels with φ(PLL) of 2%, whereas the neutral hydrogels and PLL-grafted hydrogels with higher φ(PLL) of 5% support NPC differentiation less. Gene expression of lineage markers further illustrated this trend, indicating that PLL-grafted hydrogels with an optimal φ(PLL) of 2% could be a promising cell carrier that promoted NPC functions for treatment of nerve injuries.
View details for DOI 10.1021/bm300381d
View details for Web of Science ID 000303951600049
View details for PubMedID 22533450
Lubricated Biodegradable Polymer Networks for Regulating Nerve Cell Behavior and Fabricating Nerve Conduits with a Compositional Gradient
2012; 13 (2): 358-368
We present a method of tuning surface chemistry and nerve cell behavior by photo-cross-linking methoxy poly(ethylene glycol) monoacrylate (mPEGA) with hydrophobic, semicrystalline poly(ε-caprolactone) diacrylate (PCLDA) at various weight compositions of mPEGA (ø(m)) from 2 to 30%. Improved surface wettability is achieved with corresponding decreases in friction, water contact angle, and capability of adsorbing proteins from cell culture media because of repulsive PEG chains tethered in the network. The responses of rat Schwann cell precursor line (SpL201), rat pheochromocytoma (PC12), and E14 mouse neural progenitor cells (NPCs) to the modified surfaces are evaluated. Nonmonotonic or parabolic dependence of cell attachment, spreading, proliferation, and differentiation on ø(m) is identified for these cell types with maximal values at ø(m) of 5-7%. In addition, NPCs demonstrate enhanced neuronal differentiated lineages on the mPEGA/PCLDA network at ø(m) of 5% with intermediate wettability and surface energy. This approach lays the foundation for fabricating heterogeneous nerve conduits with a compositional gradient along the wall thickness, which are able to promote nerve cell functions within the conduit while inhibiting cell attachment on the outer wall to prevent potential fibrous tissue formation following implantation.
View details for DOI 10.1021/bm201372u
View details for Web of Science ID 000300115900009
View details for PubMedID 22206477
Promoting Nerve Cell Functions on Hydrogels Grafted with Poly(L-lysine)
2012; 13 (2): 342-349
We present a novel photopolymerizable poly(L-lysine) (PLL) and use it to modify polyethylene glycol diacrylate (PEGDA) hydrogels for creating a better, permissive nerve cell niche. Compared with their neutral counterparts, these PLL-grafted hydrogels greatly enhance pheochromocytoma (PC12) cell survival in encapsulation, proliferation, and neurite growth and also promote neural progenitor cell proliferation and differentiation capacity, represented by percentages of both differentiated neurons and astrocytes. The role of efficiently controlled substrate stiffness in regulating nerve cell behavior is also investigated and a polymerizable cationic small molecule, [2-(methacryloyloxy)ethyl]-trimethylammonium chloride (MTAC), is used to compare with this newly developed PLL. The results indicate that these PLL-grafted hydrogels are promising biomaterials for nerve repair and regeneration.
View details for DOI 10.1021/bm201763n
View details for Web of Science ID 000300115900007
View details for PubMedID 22251248
Exposed hydroxyapatite particles on the surface of photo-crosslinked nanocomposites for promoting MC3T3 cell proliferation and differentiation
2011; 7 (5): 2185-2199
We present a systematic study for investigating the role of exposed hydroxyapatite (HA) nanoparticles in influencing surface characteristics and mouse pre-osteoblastic MC3T3-E1 cell behavior using nanocomposites prepared by photo-crosslinking poly(ε-caprolactone) diacrylate (PCLDA) with HA. PCLDA530 and PCLDA2000 synthesized from poly(ε-caprolactone) diol precursors with nominal molecular weights of 530 and 2000 g mol(-1) were used as the polymer matrices. Crosslinked PCLDA530 was amorphous while crosslinked PCLDA2000 was semi-crystalline. Crosslinked PCLDA/HA composites with different compositions of HA (10%, 20% and 30%) as well as crosslinked PCLDAs were characterized in terms of their composition-dependent physicochemical properties. The tensile, compressive and shear moduli were greatly enhanced by incorporating HA nanoparticles with the polymer matrices. The disk surfaces of original crosslinked PCLDA/HA nanocomposites were removed by cutting using a blade to expose HA nanoparticles that were embedded in the polymer substrates. The composition of HA was much higher on the cut surface, particularly in semi-crystalline crosslinked PCLDA2000/HA nanocomposites. The surface characteristics of original and cut crosslinked PCLDA/HA nanocomposites were compared and correlated with cell behavior on these nanocomposites. MC3T3-E1 cell attachment, proliferation and differentiation were significantly enhanced when the HA composition was increased in original crosslinked PCLDA/HA nanocomposites due to more bioactive HA, higher surface stiffness and rougher topography. More exposed HA on the surface of cut semi-crystalline PCLDA2000/HA nanocomposites resulted in improved hydrophilicity and significantly better MC3T3 cell attachment, proliferation and differentiation compared with the original surfaces. This study suggests that HA nanoparticles may not be fully exploited in polymer/HA nanocomposites where the top polymer surface covers the particles. The removal of this polymer layer can generate more desirable surfaces and osteoconductivity for bone repair and regeneration.
View details for DOI 10.1016/j.actbio.2011.01.034
View details for Web of Science ID 000290649500028
View details for PubMedID 21284960
Parabolic dependence of material properties and cell behavior on the composition of polymer networks via simultaneously controlling crosslinking density and crystallinity
2010; 31 (29): 7423-7434
A systematic investigation was performed on regulating materials properties and cell behavior using hybrid networks composed of amorphous poly(propylene fumarate) (PPF) and three poly(epsilon-caprolactone) diacrylates (PCLDAs) with variance in crystallinity and melting temperature. Through controlling both crosslinking density and crystallinity in the photo-crosslinked PPF/PCLDA blends, mechanical properties could be tuned efficiently in a wide range. For PCLDA synthesized from a low-molecular weight PCL diol precursor with a low crystallinity and a low melting point, crosslinks could completely suppress crystalline domains over the composition range in the PPF/PCLDA networks. Consequently, tensile, shear, torsional, and compression moduli all increased with the composition of PPF or the crosslinking density continuously for amorphous PPF/PCLDA networks. For PCLDAs synthesized using two PCL diols with higher molecular weights, crystallinity remained for the PCLDA compositions between approximately 80% and 100%. Minimum moduli and tensile stress at break were found at the lowest required composition of PPF for suppressing crystallinity. Surface physicochemical properties and morphology of the crosslinked blend disks have been characterized and their capabilities of adsorbing proteins from cell culture medium have been determined. Using both mouse MC3T3-E1 cells and rat Schwann cell precursor line (SpL201) cells, cell responses to these polymer networks such as cell adhesion, spreading, and proliferation were found to be dramatically distinct on different polymer networks and demonstrated non-monotonic or parabolic dependence on the network composition, coincident with the composition dependence of the mechanical properties.
View details for DOI 10.1016/j.biomaterials.2010.06.028
View details for Web of Science ID 000281183200002
View details for PubMedID 20663551
Poly(ethylene glycol)-grafted poly(propylene fumarate) networks and parabolic dependence of MC3T3 cell behavior on the network composition
2010; 31 (16): 4457-4466
We present a method to modify poly(propylene fumarate) (PPF), an injectable biomaterial for bone-tissue-engineering applications, by photo-crosslinking it with methoxy poly(ethylene glycol) monoacrylate (mPEGA) at various mPEGA compositions of 0-30%. The bulk properties such as thermal and rheological properties of uncrosslinked mPEGA/PPF blends and the mechanical properties of photo-crosslinked mPEGA/PPF blends were also investigated and correlated with surface characteristics to elaborate on the modulation of mouse MC3T3 cell adhesion, spreading, proliferation and differentiation through controlled physicochemical properties. Unlike PPF crosslinked with PEG dimethacrylate, mPEGA chains tethered on the surface of crosslinked PPF did not influence the swelling ratio in water while increased surface hydrophilicity greatly. Meanwhile, surface frictional coefficient and the capability of adsorbing proteins from cell culture medium decreased continuously with increasing the mPEGA composition in mPEGA/PPF networks. Demonstrating cell repulsive effect at the mPEGA compositions higher than 7%, the modified surfaces improved MC3T3 cell attachment, proliferation and differentiation, which reached maxima at the mPEGA composition of 5-7%. Besides revealing that mPEGA pendant chains could enhance cell responses by increasing hydrophilicity when their fraction on the hydrophobic surface was small, the present study also offered a new method of improving the wettability and performance of the scaffolds made from PPF for bone repair.
View details for DOI 10.1016/j.biomaterials.2010.02.020
View details for Web of Science ID 000277549600001
View details for PubMedID 20202682
- Poly(epsilon-caprolactone) acrylates synthesized using a facile method for fabricating networks to achieve controllable physicochemical properties and tunable cell responses POLYMER 2010; 51 (1): 164-177
- Elucidating Colorization in the Functionalization of Hydroxyl-Containing Polymers Using Unsaturated Anhydrides/Acyl Chlorides in the Presence of Triethylamine BIOMACROMOLECULES 2010; 11 (1): 304-307