My research goal is to achieve comprehensive solutions to cardiovascular clinical challenges via chemistry approaches to produce tailorable materials that serve as scaffolds or therapeutic delivery vehicles that enhance tissue regeneration. I am a trained polymer chemist with expertise in biomaterials engineering for cardiovascular regeneration and nanomedicine. My graduate research experience, under the supervision of Peter X. Ma, focused on broadening the use of tunable tissue engineering scaffolds by developing polymers with chemical functionality that can be easily and rapidly fashioned into biomimetic physical constructs and activated with regulatory signals (biomolecules, peptides, and growth factors). I accomplished this by developing novel polymer synthesis methods that are cost-effective and facile to ease the path toward clinical translation. As a postdoctoral scholar, my current training is under the co-supervision of Prof. Sarah Heilshorn and Prof. Joseph Wu as a K99/R00 MOSAIC Fellow. My work entails the development of tailored injectable hydrogels for the local delivery of therapies after a myocardial infarct.
Honors & Awards
K99/R00 Career Transition Award, Stanford University (14/07/23 - 30/06/28)
Postdoc Leadership Institute, Society for Advancement of Chicanos & Native Americans in Science (2023)
American Heart Association Postdoctoral Fellowship, Stanford University (01/01/22 - 31/01/23)
BIOX Research Mentor Award, Stanford University (2021)
Sarah Heilshorn, Postdoctoral Faculty Sponsor
Renato Navarro, Michelle Huang. Julien Roth, Kelsea Hubka, Sarah Heilshorn. "United States Patent 63/380,486 Dynamic recombinant hydrogels with degradation-independent relaxation kinetics", Leland Stanford Junior University
Renato Navarro, Peter Ma. "United States Patent 17/919,834 Biodegradable copolymers and nanofibrous scaffold thereof", Jun 15, 2023
A Library of Elastin-like Proteins with Tunable Matrix Ligands for In Vitro 3D Neural Cell Culture.
Hydrogels with encapsulated cells have widespread biomedical applications, both as tissue-mimetic 3D cultures in vitro and as tissue-engineered therapies in vivo. Within these hydrogels, the presentation of cell-instructive extracellular matrix (ECM)-derived ligands and matrix stiffness are critical factors known to influence numerous cell behaviors. While individual ECM biopolymers can be blended together to alter the presentation of cell-instructive ligands, this typically results in hydrogels with a range of mechanical properties. Synthetic systems that allow for the facile incorporation and modulation of multiple ligands without modification of matrix mechanics are highly desirable. In the present work, we leverage protein engineering to design a family of xeno-free hydrogels (i.e., devoid of animal-derived components) consisting of recombinant hyaluronan and recombinant elastin-like proteins (ELPs), cross-linked together with dynamic covalent bonds. The ELP components incorporate cell-instructive peptide ligands derived from ECM proteins, including fibronectin (RGD), laminin (IKVAV and YIGSR), collagen (DGEA), and tenascin-C (PLAEIDGIELTY and VFDNFVL). By carefully designing the protein primary sequence, we form 3D hydrogels with defined and tunable concentrations of cell-instructive ligands that have similar matrix mechanics. Utilizing this system, we demonstrate that neurite outgrowth from encapsulated embryonic dorsal root ganglion (DRG) cultures is significantly modified by cell-instructive ligand content. Thus, this library of protein-engineered hydrogels is a cell-compatible system to systematically study cell responses to matrix-derived ligands.
View details for DOI 10.1021/acs.biomac.3c00941
View details for PubMedID 37988588
Cell Microencapsulation Within Engineered Hyaluronan Elastin-Like Protein (HELP) Hydrogels.
2023; 3 (11): e917
Three-dimensional cell encapsulation has rendered itself a staple in the tissue engineering field. Using recombinantly engineered, biopolymer-based hydrogels to encapsulate cells is especially promising due to the enhanced control and tunability it affords. Here, we describe in detail the synthesis of our hyaluronan (i.e., hyaluronic acid) and elastin-like protein (HELP) hydrogel system. In addition to validating the efficacy of our synthetic process, we also demonstrate the modularity of the HELP system. Finally, we show that cells can be encapsulated within HELP gels over a range of stiffnesses, exhibit strong viability, and respond to stiffness cues. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Elastin-like protein modification with hydrazine Basic Protocol 2: Nuclear magnetic resonance quantification of elastin-like protein modification with hydrazine Basic Protocol 3: Hyaluronic acid-benzaldehyde synthesis Basic Protocol 4: Nuclear magnetic resonance quantification of hyaluronic acid-benzaldehyde Basic Protocol 5: 3D cell encapsulation in hyaluronan elastin-like protein gels.
View details for DOI 10.1002/cpz1.917
View details for PubMedID 37929691
Tunable hydrogel viscoelasticity modulates human neural maturation.
2023; 9 (42): eadh8313
Human-induced pluripotent stem cells (hiPSCs) have emerged as a promising in vitro model system for studying neurodevelopment. However, current models remain limited in their ability to incorporate tunable biomechanical signaling cues imparted by the extracellular matrix (ECM). The native brain ECM is viscoelastic and stress-relaxing, exhibiting a time-dependent response to an applied force. To recapitulate the remodelability of the neural ECM, we developed a family of protein-engineered hydrogels that exhibit tunable stress relaxation rates. hiPSC-derived neural progenitor cells (NPCs) encapsulated within these gels underwent relaxation rate-dependent maturation. Specifically, NPCs within hydrogels with faster stress relaxation rates extended longer, more complex neuritic projections, exhibited decreased metabolic activity, and expressed higher levels of genes associated with neural maturation. By inhibiting actin polymerization, we observed decreased neuritic projections and a concomitant decrease in neural maturation gene expression. Together, these results suggest that microenvironmental viscoelasticity is sufficient to bias human NPC maturation.
View details for DOI 10.1126/sciadv.adh8313
View details for PubMedID 37862423
3D printing microporous scaffolds from modular bioinks containing sacrificial, cell-encapsulating microgels.
Microgel-based biomaterials have inherent porosity and are often extrudable, making them well-suited for 3D bioprinting applications. Cells are commonly introduced into these granular inks post-printing using cell infiltration. However, due to slow cell migration speeds, this strategy struggles to achieve depth-independent cell distributions within thick 3D printed geometries. To address this, we leverage granular ink modularity by combining two microgels with distinct functions: (1) structural, UV-crosslinkable microgels made from gelatin methacryloyl (GelMA) and (2) sacrificial, cell-laden microgels made from oxidized alginate (AlgOx). We hypothesize that encapsulating cells within sacrificial AlgOx microgels would enable the simultaneous introduction of void space and release of cells at depths unachievable through cell infiltration alone. Blending the microgels in different ratios produces a family of highly printable GelMA : AlgOx microgel inks with void fractions ranging from 0.03 to 0.35. As expected, void fraction influences the morphology of human umbilical vein endothelial cells (HUVEC) within GelMA : AlgOx inks. Crucially, void fraction does not alter the ideal HUVEC distribution seen throughout the depth of 3D printed samples. This work presents a strategy for fabricating constructs with tunable porosity and depth-independent cell distribution, highlighting the promise of microgel-based inks for 3D bioprinting.
View details for DOI 10.1039/d3bm00721a
View details for PubMedID 37824082
- 3D printing microporous scaffolds from modular bioinks containing sacrificial, cell-encapsulating microgels BIOMATERIALS SCIENCE 2023
Design Parameters for Injectable Biopolymeric Hydrogels with Dynamic Covalent Chemistry Crosslinks.
Advanced healthcare materials
Dynamic covalent chemistry (DCC) crosslinks can form hydrogels with tunable mechanical properties permissive to injectability and self-healing. However, not all hydrogels with transient crosslinks are easily extrudable. For this reason, two additional design parameters must be considered when formulating DCC-crosslinked hydrogels: 1) degree of functionalization (DoF) and 2) polymer molecular weight (MW). To investigate these parameters, we formulated hydrogels comprised of two recombinant biopolymers: (1) a hyaluronic acid (HA) modified with benzaldehyde (HA-BZA) and (2) an elastin-like protein (ELP) modified with hydrazine (ELP-HYD). We synthesized several hydrogel families with distinct HA MW and DoF while keeping the ELP-HYD component constant. The resulting hydrogels had a range of stiffnesses, G' ∼ 10-1000 Pa, and extrudability, which was attributed to the combined effects of DCC crosslinks and polymer entanglements. In general, lower MW formulations required lower forces for injectability, regardless of stiffness. Higher DoF formulations exhibited more rapid self-healing. Gel extrusion through a cannula (2-m length, 0.25-mm diameter) demonstrated the potential for minimally-invasive delivery for future biomedical applications. In summary, this work highlights additional parameters that influence the injectability and network formation of DCC-crosslinked hydrogels and aims to guide future design of injectable hydrogels. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/adhm.202301265
View details for PubMedID 37389811
3D bioprinting of dynamic hydrogel bioinks enabled by small molecule modulators.
2023; 9 (13): eade7880
Three-dimensional bioprinting has emerged as a promising tool for spatially patterning cells to fabricate models of human tissue. Here, we present an engineered bioink material designed to have viscoelastic mechanical behavior, similar to that of living tissue. This viscoelastic bioink is cross-linked through dynamic covalent bonds, a reversible bond type that allows for cellular remodeling over time. Viscoelastic materials are challenging to use as inks, as one must tune the kinetics of the dynamic cross-links to allow for both extrudability and long-term stability. We overcome this challenge through the use of small molecule catalysts and competitors that temporarily modulate the cross-linking kinetics and degree of network formation. These inks were then used to print a model of breast cancer cell invasion, where the inclusion of dynamic cross-links was found to be required for the formation of invasive protrusions. Together, we demonstrate the power of engineered, dynamic bioinks to recapitulate the native cellular microenvironment for disease modeling.
View details for DOI 10.1126/sciadv.ade7880
View details for PubMedID 37000873
Elastin-like protein hydrogels with controllable stress relaxation rate and stiffness modulate endothelial cell function.
Journal of biomedical materials research. Part A
Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell-applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin-like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine-modified ELP (ELP-HYD) and aldehyde/benzaldehyde-modified polyethylene glycol (PEG-ALD/PEG-BZA). The reversible DCC crosslinks in ELP-PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast-relaxing or slow-relaxing hydrogels with a range of stiffness (500-3300Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two-dimensional substrates, on which ECs exhibited greater cell spreading on fast-relaxing hydrogels up through 3days, compared with slow-relaxing hydrogels at the same stiffness. In three-dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast-relaxing, low-stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast-relaxing, low-stiffness hydrogel produced significantly more vascularization compared with the slow-relaxing, low-stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast-relaxing, low-stiffness hydrogels supported the highest capillary density in vivo.
View details for DOI 10.1002/jbm.a.37520
View details for PubMedID 36861665
Tuning Polymer Hydrophilicity to Regulate Gel Mechanics and Encapsulated Cell Morphology.
Advanced healthcare materials
Mechanically tunable hydrogels are attractive platforms for three-dimensional cell culture, as hydrogel stiffness plays an important role in cell behavior. Traditionally, hydrogel stiffness has been controlled through altering either the polymer concentration or the stoichiometry between crosslinker reactive groups. Here, we present an alternative strategy based upon tuning the hydrophilicity of an elastin-like protein (ELP). ELPs undergo a phase transition that leads to protein aggregation at increasing temperatures. We hypothesize that increasing this transition temperature through bioconjugation with azide-containing molecules of increasing hydrophilicity will allow direct control of the resulting gel stiffness by making the crosslinking groups more accessible. These azide-modified ELPs are crosslinked into hydrogels with bicyclononyne-modified hyaluronic acid (HA-BCN) using bioorthogonal, click chemistry, resulting in hydrogels with tunable storage moduli (100-1000Pa). Human mesenchymal stromal cells, human umbilical vein endothelial cells, and human neural progenitor cells are all observed to alter their cell morphology when encapsulated within hydrogels of varying stiffness. Taken together, we demonstrate the use of protein hydrophilicity as a lever to tune hydrogel mechanical properties. These hydrogels have tunable moduli over a stiffness range relevant to soft tissues, support the viability of encapsulated cells, and modify cell spreading as a consequence of gel stiffness. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/adhm.202200011
View details for PubMedID 35373510