Publications by authors named "Jordan S Miller"

32 Publications

Contextual cues from cancer cells govern cancer-associated fibroblast heterogeneity.

Cell Rep 2021 Apr;35(3):109009

Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis.
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http://dx.doi.org/10.1016/j.celrep.2021.109009DOI Listing
April 2021

Development, characterization, and applications of multi-material stereolithography bioprinting.

Sci Rep 2021 Feb 4;11(1):3171. Epub 2021 Feb 4.

Department of Bioengineering, Rice University, Houston, TX, USA.

As a 3D bioprinting technique, hydrogel stereolithography has historically been limited in its ability to capture the spatial heterogeneity that permeates mammalian tissues and dictates structure-function relationships. This limitation stems directly from the difficulty of preventing unwanted material mixing when switching between different liquid bioinks. Accordingly, we present the development, characterization, and application of a multi-material stereolithography bioprinter that provides controlled material selection, yields precise regional feature alignment, and minimizes bioink mixing. Fluorescent tracers were first used to highlight the broad design freedoms afforded by this fabrication strategy, complemented by morphometric image analysis to validate architectural fidelity. To evaluate the bioactivity of printed gels, 344SQ lung adenocarcinoma cells were printed in a 3D core/shell architecture. These cells exhibited native phenotypic behavior as evidenced by apparent proliferation and formation of spherical multicellular aggregates. Cells were also printed as pre-formed multicellular aggregates, which appropriately developed invasive protrusions in response to hTGF-β1. Finally, we constructed a simplified model of intratumoral heterogeneity with two separate sub-populations of 344SQ cells, which together grew over 14 days to form a dense regional interface. Together, these studies highlight the potential of multi-material stereolithography to probe heterotypic interactions between distinct cell types in tissue-specific microenvironments.
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http://dx.doi.org/10.1038/s41598-021-82102-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862383PMC
February 2021

Thermofluidic heat exchangers for actuation of transcription in artificial tissues.

Sci Adv 2020 Sep 30;6(40). Epub 2020 Sep 30.

Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.

Spatial patterns of gene expression in living organisms orchestrate cell decisions in development, homeostasis, and disease. However, most methods for reconstructing gene patterning in 3D cell culture and artificial tissues are restricted by patterning depth and scale. We introduce a depth- and scale-flexible method to direct volumetric gene expression patterning in 3D artificial tissues, which we call "heat exchangers for actuation of transcription" (HEAT). This approach leverages fluid-based heat transfer from printed networks in the tissues to activate heat-inducible transgenes expressed by embedded cells. We show that gene expression patterning can be tuned both spatially and dynamically by varying channel network architecture, fluid temperature, fluid flow direction, and stimulation timing in a user-defined manner and maintained in vivo. We apply this approach to activate the 3D positional expression of Wnt ligands and Wnt/β-catenin pathway regulators, which are major regulators of development, homeostasis, regeneration, and cancer throughout the animal kingdom.
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http://dx.doi.org/10.1126/sciadv.abb9062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527231PMC
September 2020

Generation of model tissues with dendritic vascular networks via sacrificial laser-sintered carbohydrate templates.

Nat Biomed Eng 2020 09 29;4(9):916-932. Epub 2020 Jun 29.

Department of Bioengineering, Rice University, Houston, TX, USA.

Sacrificial templates for patterning perfusable vascular networks in engineered tissues have been constrained in architectural complexity, owing to the limitations of extrusion-based 3D printing techniques. Here, we show that cell-laden hydrogels can be patterned with algorithmically generated dendritic vessel networks and other complex hierarchical networks by using sacrificial templates made from laser-sintered carbohydrate powders. We quantified and modulated gradients of cell proliferation and cell metabolism emerging in response to fluid convection through these networks and to diffusion of oxygen and metabolites out of them. We also show scalable strategies for the fabrication, perfusion culture and volumetric analysis of large tissue-like constructs with complex and heterogeneous internal vascular architectures. Perfusable dendritic networks in cell-laden hydrogels may help sustain thick and densely cellularized engineered tissues, and assist interrogations of the interplay between mass transport and tissue function.
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http://dx.doi.org/10.1038/s41551-020-0566-1DOI Listing
September 2020

Disturbed flow disrupts the blood-brain barrier in a 3D bifurcation model.

Biofabrication 2020 02 27;12(2):025020. Epub 2020 Feb 27.

Department of Biomedical Engineering, Rowan University, Glassboro, NJ 08028, United States of America.

The effect of disturbed flow profiles on the endothelium have been studied extensively in systemic vasculature, but less is known about the response of the blood-brain barrier (BBB) to these flow regimes. Here we investigate the effect of disturbed flow on the integrity of the BBB using a three-dimensional, perfusable bifurcation model consisting of a co-culture of endothelial cells with mural and glial cells. Experimental flow patterns predicted by computational fluid dynamics mimic in vivo flow regimes, specifically the presence of a recirculation zone immediately downstream of the bifurcation. Dextran permeability assays and immunostaining with markers for tight junctions show that barrier disruption is significantly greater in areas of disturbed flow compared to fully developed regions downstream of the bifurcation. Probing crosstalk between cell types suggests that disturbed flow causes barrier breakdown independent of endothelial-mural and endothelial-glial interaction. Overall, disturbed flow-induced disruption of the blood-brain barrier suggests that flow-mediated mechanisms may contribute to vascular pathologies in the central nervous system.
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http://dx.doi.org/10.1088/1758-5090/ab5898DOI Listing
February 2020

Multivascular networks and functional intravascular topologies within biocompatible hydrogels.

Science 2019 05;364(6439):458-464

Department of Bioengineering, Rice University, Houston, TX 77005, USA.

Solid organs transport fluids through distinct vascular networks that are biophysically and biochemically entangled, creating complex three-dimensional (3D) transport regimes that have remained difficult to produce and study. We establish intravascular and multivascular design freedoms with photopolymerizable hydrogels by using food dye additives as biocompatible yet potent photoabsorbers for projection stereolithography. We demonstrate monolithic transparent hydrogels, produced in minutes, comprising efficient intravascular 3D fluid mixers and functional bicuspid valves. We further elaborate entangled vascular networks from space-filling mathematical topologies and explore the oxygenation and flow of human red blood cells during tidal ventilation and distension of a proximate airway. In addition, we deploy structured biodegradable hydrogel carriers in a rodent model of chronic liver injury to highlight the potential translational utility of this materials innovation.
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http://dx.doi.org/10.1126/science.aav9750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769170PMC
May 2019

A novel ex vivo tumor system identifies Src-mediated invasion and metastasis in mesenchymal tumor cells in non-small cell lung cancer.

Sci Rep 2019 03 20;9(1):4819. Epub 2019 Mar 20.

Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Lung cancer is the foremost cause of cancer related deaths in the U.S. It is a heterogeneous disease composed of genetically and phenotypically distinct tumor cells surrounded by heterotypic cells and extracellular matrix dynamically interacting with the tumor cells. Research in lung cancer is often restricted to patient-derived tumor specimens, in vitro cell cultures and limited animal models, which fail to capture the cellular or microenvironment heterogeneity of the tumor. Therefore, our knowledge is primarily focused on cancer-cell autonomous aberrations. For a fundamental understanding of lung cancer progression and an exploration of therapeutic options, we focused our efforts to develop an Ex Vivo Tumor platform to culture tumors in 3D matrices, which retains tumor cell heterogeneity arising due to in vivo selection pressure and environmental influences and recapitulate responses of tumor cells to external manipulations. To establish this model, implanted syngeneic murine tumors from a mutant KRAS/p53 model were harvested to yield multicellular tumor aggregates followed by culture in 3D extracellular matrices. Using this system, we identified Src signaling as an important driver of invasion and metastasis in lung cancer and demonstrate that EVTs are a robust experimental tool bridging the gap between conventional in vitro and in vivo models.
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http://dx.doi.org/10.1038/s41598-019-41301-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6427036PMC
March 2019

3D bioprinting: improving in vitro models of metastasis with heterogeneous tumor microenvironments.

Dis Model Mech 2017 01;10(1):3-14

Department of Bioengineering, Rice University, Houston, TX 77005, USA

Even with many advances in treatment over the past decades, cancer still remains a leading cause of death worldwide. Despite the recognized relationship between metastasis and increased mortality rate, surprisingly little is known about the exact mechanism of metastatic progression. Currently available in vitro models cannot replicate the three-dimensionality and heterogeneity of the tumor microenvironment sufficiently to recapitulate many of the known characteristics of tumors in vivo Our understanding of metastatic progression would thus be boosted by the development of in vitro models that could more completely capture the salient features of cancer biology. Bioengineering groups have been working for over two decades to create in vitro microenvironments for application in regenerative medicine and tissue engineering. Over this time, advances in 3D printing technology and biomaterials research have jointly led to the creation of 3D bioprinting, which has improved our ability to develop in vitro models with complexity approaching that of the in vivo tumor microenvironment. In this Review, we give an overview of 3D bioprinting methods developed for tissue engineering, which can be directly applied to constructing in vitro models of heterogeneous tumor microenvironments. We discuss considerations and limitations associated with 3D printing and highlight how these advances could be harnessed to better model metastasis and potentially guide the development of anti-cancer strategies.
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http://dx.doi.org/10.1242/dmm.025049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5278522PMC
January 2017

Editorial: Special Issue on 3D Printing of Biomaterials.

ACS Biomater Sci Eng 2016 Oct;2(10):1658-1661

Department of Bioengineering, University of Pennsylvania.

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http://dx.doi.org/10.1021/acsbiomaterials.6b00566DOI Listing
October 2016

3D-printed fluidic networks as vasculature for engineered tissue.

Lab Chip 2016 05;16(11):2025-43

Department of Bioengineering, Rice University, Houston, TX, USA.

Fabrication of vascular networks within engineered tissue remains one of the greatest challenges facing the fields of biomaterials and tissue engineering. Historically, the structural complexity of vascular networks has limited their fabrication in tissues engineered in vitro. Recently, however, key advances have been made in constructing fluidic networks within biomaterials, suggesting a strategy for fabricating the architecture of the vasculature. These techniques build on emerging technologies within the microfluidics community as well as on 3D printing. The freeform fabrication capabilities of 3D printing are allowing investigators to fabricate fluidic networks with complex architecture inside biomaterial matrices. In this review, we examine the most exciting 3D printing-based techniques in this area. We also discuss opportunities for using these techniques to address open questions in vascular biology and biophysics, as well as for engineering therapeutic tissue substitutes in vitro.
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http://dx.doi.org/10.1039/c6lc00193aDOI Listing
May 2016

Ultrahigh-throughput Generation and Characterization of Cellular Aggregates in Laser-ablated Microwells of Poly(dimethylsiloxane).

RSC Adv 2016 Jan 12;6(11):8980-8991. Epub 2016 Jan 12.

Department of Bioengineering, Rice University, Houston, Texas, USA.

Aggregates of cells, also known as multicellular aggregates (MCAs), have been used as microscale tissues in the fields of cancer biology, regenerative medicine, and developmental biology for many decades. However, small MCAs (fewer than 100 cells per aggregate) have remained challenging to manufacture in large quantities at high uniformity. Forced aggregation into microwells offers a promising solution for forming consistent aggregates, but commercial sources of microwells are expensive, complicated to manufacture, or lack the surface packing densities that would significantly improve MCA production. To address these concerns, we custom-modified a commercial laser cutter to provide complete control over laser ablation and directly generate microwells in a poly(dimethylsiloxane) (PDMS) substrate. We achieved ultra rapid microwell production speeds (>50,000 microwells/hr) at high areal packing densities (1,800 microwells/cm) and over large surface areas for cell culture (60 cm). Variation of the PDMS substrate distance from the laser focal plane during ablation allowed for the generation of microwells with a variety of sizes, contours, and aspect ratios. Casting of high-fidelity microneedle masters in polyurethane allowed for non-ablative microwell reproduction through replica molding. MCAs of human bone marrow derived mesenchymal stem cells (hMSCs), murine 344SQ metastatic adenocarcinoma cells, and human C4-2 prostate cancer cells were generated in our system with high uniformity within 24 hours, and computer vision software aided in the ultra-high-throughput analysis of harvested aggregates. Moreover, MCAs maintained invasive capabilities in 3D migration assays. In particular, 344SQ MCAs demonstrated epithelial lumen formation on Matrigel, and underwent EMT and invasion in the presence of TGF-β. We expect this technique to find broad utility in the generation and cultivation of cancer cell aggregates, primary cell aggregates, and embryoid bodies.
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http://dx.doi.org/10.1039/C5RA26022ADOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4792302PMC
January 2016

Open-Source Selective Laser Sintering (OpenSLS) of Nylon and Biocompatible Polycaprolactone.

PLoS One 2016 3;11(2):e0147399. Epub 2016 Feb 3.

Department of Bioengineering, Rice University, Houston, Texas, United States of America.

Selective Laser Sintering (SLS) is an additive manufacturing process that uses a laser to fuse powdered starting materials into solid 3D structures. Despite the potential for fabrication of complex, high-resolution structures with SLS using diverse starting materials (including biomaterials), prohibitive costs of commercial SLS systems have hindered the wide adoption of this technology in the scientific community. Here, we developed a low-cost, open-source SLS system (OpenSLS) and demonstrated its capacity to fabricate structures in nylon with sub-millimeter features and overhanging regions. Subsequently, we demonstrated fabrication of polycaprolactone (PCL) into macroporous structures such as a diamond lattice. Widespread interest in using PCL for bone tissue engineering suggests that PCL lattices are relevant model scaffold geometries for engineering bone. SLS of materials with large powder grain size (~500 μm) leads to part surfaces with high roughness, so we further introduced a simple vapor-smoothing technique to reduce the surface roughness of sintered PCL structures which further improves their elastic modulus and yield stress. Vapor-smoothed PCL can also be used for sacrificial templating of perfusable fluidic networks within orthogonal materials such as poly(dimethylsiloxane) silicone. Finally, we demonstrated that human mesenchymal stem cells were able to adhere, survive, and differentiate down an osteogenic lineage on sintered and smoothed PCL surfaces, suggesting that OpenSLS has the potential to produce PCL scaffolds useful for cell studies. OpenSLS provides the scientific community with an accessible platform for the study of laser sintering and the fabrication of complex geometries in diverse materials.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147399PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4739701PMC
July 2016

Three dimensional model for surgical planning in resection of thoracic tumors.

Int J Surg Case Rep 2015 3;16:127-9. Epub 2015 Oct 3.

Department of Bioengineering, Rice University, Houston, TX, United States.

Introduction: The computed tomography scan provides vital information about the relationship of thoracic malignancies to the surrounding structures and aids in surgical planning. However, it can be difficult to visualize the images in a two-dimensional screen to interpret the full extent of the relationship between important structures in the surgical field.

Presentation Of Case: We report two cases where we used a three-dimensional printed model to aid in the surgical resection of thoracic malignancies.

Discussion: Careful planning is necessary to resect thoracic malignancies. Although two-dimensional images of the thoracic malignancies provide vital information about the tumor and its surrounding structures, the three-dimensional printed model can provide more accurate information about the tumor and assist in surgical planning.

Conclusion: Three-dimensional printed model provide better visualization of complex thoracic tumors, aid in counseling the patient about the surgical procedure and assisted in surgical resection of thoracic malignancy.
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http://dx.doi.org/10.1016/j.ijscr.2015.09.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643475PMC
November 2015

In Vivo Anastomosis and Perfusion of a Three-Dimensionally-Printed Construct Containing Microchannel Networks.

Tissue Eng Part C Methods 2016 Jan 14;22(1):1-7. Epub 2015 Dec 14.

1 Division of Cardiovascular Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania , Philadelphia, Pennsylvania.

The field of tissue engineering has advanced the development of increasingly biocompatible materials to mimic the extracellular matrix of vascularized tissue. However, a majority of studies instead rely on a multiday inosculation between engineered vessels and host vasculature rather than the direct connection of engineered microvascular networks with host vasculature. We have previously demonstrated that the rapid casting of three-dimensionally-printed (3D) sacrificial carbohydrate glass is an expeditious and a reliable method of creating scaffolds with 3D microvessel networks. Here, we describe a new surgical technique to directly connect host femoral arteries to patterned microvessel networks. Vessel networks were connected in vivo in a rat femoral artery graft model. We utilized laser Doppler imaging to monitor hind limb ischemia for several hours after implantation and thus measured the vascular patency of implants that were anastomosed to the femoral artery. This study may provide a method to overcome the challenge of rapid oxygen and nutrient delivery to engineered vascularized tissues implanted in vivo.
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http://dx.doi.org/10.1089/ten.TEC.2015.0239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722541PMC
January 2016

Degradable hydrogels derived from PEG-diacrylamide for hepatic tissue engineering.

J Biomed Mater Res A 2015 Oct 30;103(10):3331-8. Epub 2015 Apr 30.

Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139.

Engineered tissue constructs have the potential to augment or replace whole organ transplantation for the treatment of liver failure. Poly(ethylene glycol) (PEG)-based systems are particularly promising for the construction of engineered liver tissue due to their biocompatibility and amenability to modular addition of bioactive factors. To date, primary hepatocytes have been successfully encapsulated in non-degradable hydrogels based on PEG-diacrylate (PEGDA). In this study, we describe a hydrogel system based on PEG-diacrylamide (PEGDAAm) containing matrix-metalloproteinase sensitive (MMP-sensitive) peptide in the hydrogel backbone that is suitable for hepatocyte culture both in vitro and after implantation. By replacing hydrolytically unstable esters in PEGDA with amides in PEGDAAm, resultant hydrogels resisted non-specific hydrolysis, while still allowing for MMP-mediated hydrogel degradation. Optimization of polymerization conditions, hepatocellular density, and multicellular tissue composition modulated both the magnitude and longevity of hepatic function in vitro. Importantly, hepatic PEGDAAm-based tissues survived and functioned for over 3 weeks after implantation ectopically in the intraperitoneal (IP) space of nude mice. Together, these studies suggest that MMP-sensitive PEGDAAm-based hydrogels may be a useful material system for applications in tissue engineering and regenerative medicine. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3331-3338, 2015.
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http://dx.doi.org/10.1002/jbm.a.35478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4890565PMC
October 2015

Open-source three-dimensional printing of biodegradable polymer scaffolds for tissue engineering.

J Biomed Mater Res A 2014 Dec;102(12):4326-35

The fabrication of scaffolds for tissue engineering requires elements of customization depending on the application and is often limited due to the flexibility of the processing technique. This investigation seeks to address this obstacle by utilizing an open-source three-dimensional printing (3DP) system that allows vast customizability and facilitates reproduction of experiments. The effects of processing parameters on printed poly(ε-caprolactone) scaffolds with uniform and gradient pore architectures have been characterized with respect to fiber and pore morphology and mechanical properties. The results demonstrate the ability to tailor the fiber diameter, pore size, and porosity through modification of pressure, printing speed, and programmed fiber spacing. A model was also used to predict the compressive mechanical properties of uniform and gradient scaffolds, and it was found that modulus and yield strength declined with increasing porosity. The use of open-source 3DP technologies for printing tissue-engineering scaffolds provides a flexible system that can be readily modified at a low cost and is supported by community documentation. In this manner, the 3DP system is more accessible to the scientific community, which further facilitates the translation of these technologies toward successful tissue-engineering strategies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4266185PMC
http://dx.doi.org/10.1002/jbm.a.35108DOI Listing
December 2014

Tissue-engineered, hydrogel-based endothelial progenitor cell therapy robustly revascularizes ischemic myocardium and preserves ventricular function.

J Thorac Cardiovasc Surg 2014 Sep 28;148(3):1090-7; discussion 1097-8. Epub 2014 Jun 28.

Department of Cardiothoracic Surgery, Stanford University, Stanford, Calif. Electronic address:

Objectives: Cell-based angiogenic therapy for ischemic heart failure has had limited clinical impact, likely related to low cell retention (<1%) and dispersion. We developed a novel, tissue-engineered, hydrogel-based cell-delivery strategy to overcome these limitations and provide prolonged regional retention of myocardial endothelial progenitor cells at high cell dosage.

Methods: Endothelial progenitor cells were isolated from Wistar rats and encapsulated in fibrin gels. In vitro viability was quantified using a fluorescent live-dead stain of transgenic enhanced green fluorescent protein(+) endothelial progenitor cells. Endothelial progenitor cell-laden constructs were implanted onto ischemic rat myocardium in a model of acute myocardial infarction (left anterior descending ligation) for 4 weeks. Intramyocardial cell injection (2 × 10(6) endothelial progenitor cells), empty fibrin, and isolated left anterior descending ligation groups served as controls. Hemodynamics were quantified using echocardiography, Doppler flow analysis, and intraventricular pressure-volume analysis. Vasculogenesis and ventricular geometry were quantified. Endothelial progenitor cell migration was analyzed by using endothelial progenitor cells from transgenic enhanced green fluorescent protein(+) rodents.

Results: Endothelial progenitor cells demonstrated an overall 88.7% viability for all matrix and cell conditions investigated after 48 hours. Histologic assessment of 1-week implants demonstrated significant migration of transgenic enhanced green fluorescent protein(+) endothelial progenitor cells from the fibrin matrix to the infarcted myocardium compared with intramyocardial cell injection (28 ± 12.3 cells/high power field vs 2.4 ± 2.1 cells/high power field, P = .0001). We also observed a marked increase in vasculogenesis at the implant site. Significant improvements in ventricular hemodynamics and geometry were present after endothelial progenitor cell-hydrogel therapy compared with control.

Conclusions: We present a tissue-engineered, hydrogel-based endothelial progenitor cell-mediated therapy to enhance cell delivery, cell retention, vasculogenesis, and preservation of myocardial structure and function.
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http://dx.doi.org/10.1016/j.jtcvs.2014.06.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4155940PMC
September 2014

The billion cell construct: will three-dimensional printing get us there?

Authors:
Jordan S Miller

PLoS Biol 2014 Jun 17;12(6):e1001882. Epub 2014 Jun 17.

Department of Bioengineering, Rice University, Houston, Texas, United States of America.

How structure relates to function--across spatial scales, from the single molecule to the whole organism--is a central theme in biology. Bioengineers, however, wrestle with the converse question: will function follow form? That is, we struggle to approximate the architecture of living tissues experimentally, hoping that the structure we create will lead to the function we desire. A new means to explore the relationship between form and function in living tissue has arrived with three-dimensional printing, but the technology is not without limitations.
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http://dx.doi.org/10.1371/journal.pbio.1001882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4061004PMC
June 2014

Engineering Escherichia coli for light-activated cytolysis of mammalian cells.

ACS Synth Biol 2014 Dec 14;3(12):944-8. Epub 2014 Jan 14.

Department of Bioengineering, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States of America.

By delivering payloads in response to specific exogenous stimuli, smart bacterial therapeutics have the potential to overcome many limitations of conventional therapies, including poor targeting specificity and dosage control in current cancer treatments. Although not yet explored as a trigger for bacterial drug delivery, light is an ideal induction mechanism because it offers fine spatiotemporal control and is easily and safely administered. Using recent advances in optogenetics, we have engineered two strains of Escherichia coli to secrete a potent mammalian cytotoxin in response to blue or red light. The tools in this study demonstrate the initial feasibility of light-activated bacterial therapeutics for applications such as tumor cytolysis, and their modular nature should enable simple substitution of other payloads of interest.
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http://dx.doi.org/10.1021/sb400174sDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264543PMC
December 2014

Geometric control of vascular networks to enhance engineered tissue integration and function.

Proc Natl Acad Sci U S A 2013 May 22;110(19):7586-91. Epub 2013 Apr 22.

Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA 19104, USA.

Tissue vascularization and integration with host circulation remains a key barrier to the translation of engineered tissues into clinically relevant therapies. Here, we used a microtissue molding approach to demonstrate that constructs containing highly aligned "cords" of endothelial cells triggered the formation of new capillaries along the length of the patterned cords. These vessels became perfused with host blood as early as 3 d post implantation and became progressively more mature through 28 d. Immunohistochemical analysis showed that the neovessels were composed of human and mouse endothelial cells and exhibited a mature phenotype, as indicated by the presence of alpha-smooth muscle actin-positive pericytes. Implantation of cords with a prescribed geometry demonstrated that they provided a template that defined the neovascular architecture in vivo. To explore the utility of this geometric control, we implanted primary rat and human hepatocyte constructs containing randomly organized endothelial networks vs. ordered cords. We found substantially enhanced hepatic survival and function in the constructs containing ordered cords following transplantation in mice. These findings demonstrate the importance of multicellular architecture in tissue integration and function, and our approach provides a unique strategy to engineer vascular architecture.
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http://dx.doi.org/10.1073/pnas.1217796110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651499PMC
May 2013

Multidimensional traction force microscopy reveals out-of-plane rotational moments about focal adhesions.

Proc Natl Acad Sci U S A 2013 Jan 31;110(3):881-6. Epub 2012 Dec 31.

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.

Recent methods have revealed that cells on planar substrates exert both shear (in-plane) and normal (out-of-plane) tractions against the extracellular matrix (ECM). However, the location and origin of the normal tractions with respect to the adhesive and cytoskeletal elements of cells have not been elucidated. We developed a high-spatiotemporal-resolution, multidimensional (2.5D) traction force microscopy to measure and model the full 3D nature of cellular forces on planar 2D surfaces. We show that shear tractions are centered under elongated focal adhesions whereas upward and downward normal tractions are detected on distal (toward the cell edge) and proximal (toward the cell body) ends of adhesions, respectively. Together, these forces produce significant rotational moments about focal adhesions in both protruding and retracting peripheral regions. Temporal 2.5D traction force microscopy analysis of migrating and spreading cells shows that these rotational moments are highly dynamic, propagating outward with the leading edge of the cell. Finally, we developed a finite element model to examine how rotational moments could be generated about focal adhesions in a thin lamella. Our model suggests that rotational moments can be generated largely via shear lag transfer to the underlying ECM from actomyosin contractility applied at the intracellular surface of a rigid adhesion of finite thickness. Together, these data demonstrate and probe the origin of a previously unappreciated multidimensional stress profile associated with adhesions and highlight the importance of new approaches to characterize cellular forces.
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http://dx.doi.org/10.1073/pnas.1207997110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549134PMC
January 2013

Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues.

Nat Mater 2012 Sep 1;11(9):768-74. Epub 2012 Jul 1.

Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

In the absence of perfusable vascular networks, three-dimensional (3D) engineered tissues densely populated with cells quickly develop a necrotic core. Yet the lack of a general approach to rapidly construct such networks remains a major challenge for 3D tissue culture. Here, we printed rigid 3D filament networks of carbohydrate glass, and used them as a cytocompatible sacrificial template in engineered tissues containing living cells to generate cylindrical networks that could be lined with endothelial cells and perfused with blood under high-pressure pulsatile flow. Because this simple vascular casting approach allows independent control of network geometry, endothelialization and extravascular tissue, it is compatible with a wide variety of cell types, synthetic and natural extracellular matrices, and crosslinking strategies. We also demonstrated that the perfused vascular channels sustained the metabolic function of primary rat hepatocytes in engineered tissue constructs that otherwise exhibited suppressed function in their core.
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http://dx.doi.org/10.1038/nmat3357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3586565PMC
September 2012

Bioresponsive mesoporous silica nanoparticles for triggered drug release.

J Am Chem Soc 2011 Dec 18;133(49):19582-5. Epub 2011 Nov 18.

Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute Technology, Cambridge, Massachusetts 02139, USA.

Mesoporous silica nanoparticles (MSNPs) have garnered a great deal of attention as potential carriers for therapeutic payloads. However, achieving triggered drug release from MSNPs in vivo has been challenging. Here, we describe the synthesis of stimulus-responsive polymer-coated MSNPs and the loading of therapeutics into both the core and shell domains. We characterize MSNP drug-eluting properties in vitro and demonstrate that the polymer-coated MSNPs release doxorubicin in response to proteases present at a tumor site in vivo, resulting in cellular apoptosis. These results demonstrate the utility of polymer-coated nanoparticles in specifically delivering an antitumor payload.
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http://dx.doi.org/10.1021/ja206998xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3295203PMC
December 2011

Fabrication of Multifaceted Micropatterned Surfaces with Laser Scanning Lithography.

Adv Funct Mater 2011 Aug 31;21(15):2876-2888. Epub 2011 May 31.

Department of Bioengineering, Rice University, Houston, Texas 77005 (USA).

The implementation of engineered surfaces presenting micrometer-sized patterns of cell adhesive ligands against a biologically inert background has led to numerous discoveries in fundamental cell biology. While existing surface patterning strategies allow for pattering of a single ligand it is still challenging to fabricate surfaces displaying multiple patterned ligands. To address this issue we implemented Laser Scanning Lithography (LSL), a laser-based thermal desorption technique, to fabricate multifaceted, micropatterned surfaces that display independent arrays of subcellular-sized patterns of multiple adhesive ligands with each ligand confined to its own array. We demonstrate that LSL is a highly versatile "maskless" surface patterning strategy that provides the ability to create patterns with features ranging from 450 nm to 100 μm, topography ranging from -1 to 17 nm, and to fabricate both stepwise and smooth ligand surface density gradients. As validation for their use in cell studies, surfaces presenting orthogonally interwoven arrays of 1×8 μm elliptical patterns of Gly-Arg-Gly-Asp-terminated alkanethiol self-assembled monolayers and human plasma fibronectin are produced. Human umbilical vein endothelial cells cultured on these multifaceted surfaces form adhesion sites to both ligands simultaneously and utilize both ligands for lamella formation during migration. The ability to create multifaceted, patterned surfaces with tight control over pattern size, spacing, and topography provides a platform to simultaneously investigate the complex interactions of extracellular matrix geometry, biochemistry, and topography on cell adhesion and downstream cell behavior.
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http://dx.doi.org/10.1002/adfm.201100297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978433PMC
August 2011

Decreased cell adhesion promotes angiogenesis in a Pyk2-dependent manner.

Exp Cell Res 2011 Aug 18;317(13):1860-71. Epub 2011 May 18.

Department of Bioengineering, University of Pennsylvania, 210 South 33rd Street, Philadelphia, PA 19104, USA.

Angiogenesis is regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Here, we show that angiogenic sprouting in natural and engineered three-dimensional matrices exhibited a biphasic response, with peak sprouting when adhesion to the matrix was limited to intermediate levels. Examining changes in global gene expression to determine a genetic basis for this response, we demonstrate a vascular endothelial growth factor (VEGF)-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was limited, whereas cells on highly adhesive surfaces upregulated genes associated with proliferation. To explore a mechanistic basis for this effect, we turned to focal adhesion kinase (FAK), a central player in adhesion signaling previously implicated in angiogenesis, and its homologue, proline-rich tyrosine kinase 2 (Pyk2). While FAK signaling had some impact, our results suggested that Pyk2 can regulate both gene expression and endothelial sprouting through its enhanced activation by VEGF in limited adhesion contexts. We also demonstrate decreased sprouting of tissue explants from Pyk2-null mice as compared to wild type mice as further confirmation of the role of Pyk2 in angiogenic sprouting. These results suggest a surprising finding that limited cell adhesion can enhance endothelial responsiveness to VEGF and demonstrate a novel role for Pyk2 in the adhesive regulation of angiogenesis.
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http://dx.doi.org/10.1016/j.yexcr.2011.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3123418PMC
August 2011

Measurement of mechanical tractions exerted by cells in three-dimensional matrices.

Nat Methods 2010 Dec 14;7(12):969-71. Epub 2010 Nov 14.

Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Quantitative measurements of cell-generated forces have heretofore required that cells be cultured on two-dimensional substrates. We describe a technique to quantitatively measure three-dimensional traction forces exerted by cells fully encapsulated in well-defined elastic hydrogel matrices. Using this approach we measured traction forces for several cell types in various contexts and revealed patterns of force generation attributable to morphologically distinct regions of cells as they extend into the surrounding matrix.
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http://dx.doi.org/10.1038/nmeth.1531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056435PMC
December 2010

Multilayer microfluidic PEGDA hydrogels.

Biomaterials 2010 Jul 5;31(21):5491-7. Epub 2010 May 5.

Department of Bioengineering, Rice University, 6100 Main Street, Houston, TX 77005, United States.

Development of robust 3D tissue analogs in vitro is limited by passive, diffusional mass transport. Perfused microfluidic tissue engineering scaffolds hold the promise to improve mass transport limitations and promote the development of complex, metabolically dense, and clinically relevant tissues. We report a simple and robust multilayer replica molding technique in which poly(dimethylsiloxane) (PDMS) and poly(ethylene glycol) diacrylate (PEGDA) are serially replica molded to develop microfluidic PEGDA hydrogel networks embedded within independently fabricated PDMS housings. We demonstrate the ability to control solute-scaffold effective diffusivity as a function of solute molecular weight and hydrogel concentration. Within cell laden microfluidic hydrogels, we demonstrate increased cellular viability in perfused hydrogel systems compared to static controls. We observed a significant increase in cell viability at all time points greater than zero at distances up to 1 mm from the perfused channel. Knowledge of spatiotemporal mass transport and cell viability gradients provides useful engineering design parameters necessary to maximize overall scaffold viability and metabolic density. This work has applications in the development of hydrogels as in vitro diagnostics and ultimately as regenerative medicine based therapeutics.
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http://dx.doi.org/10.1016/j.biomaterials.2010.03.031DOI Listing
July 2010

Bioactive hydrogels made from step-growth derived PEG-peptide macromers.

Biomaterials 2010 May 6;31(13):3736-43. Epub 2010 Feb 6.

Department of Bioengineering, University of Pennsylvania, 210 S. 33rd St., 510 Skirkanich Hall, Philadelphia, PA 19104, USA.

Synthetic hydrogels based on poly(ethylene glycol) (PEG) have been used as biomaterials for cell biology and tissue engineering investigations. Bioactive PEG-based gels have largely relied on heterobifunctional or multi-arm PEG precursors that can be difficult to synthesize and characterize or expensive to obtain. Here, we report an alternative strategy, which instead uses inexpensive and readily available PEG precursors to simplify reactant sourcing. This new approach provides a robust system in which to probe cellular interactions with the microenvironment. We used the step-growth polymerization of PEG diacrylate (PEGDA, 3400Da) with bis-cysteine matrix metalloproteinase (MMP)-sensitive peptides via Michael-type addition to form biodegradable photoactive macromers of the form acrylate-PEG-(peptide-PEG)(m)-acrylate. The molecular weight (MW) of these macromers is controlled by the stoichiometry of the reaction, with a high proportion of resultant macromer species greater than 500kDa. In addition, the polydispersity of these materials was nearly identical for three different MMP-sensitive peptide sequences subjected to the same reaction conditions. When photopolymerized into hydrogels, these high MW materials exhibit increased swelling and sensitivity to collagenase-mediated degradation as compared to previously published PEG hydrogel systems. Cell-adhesive acrylate-PEG-CGRGDS was synthesized similarly and its immobilization and stability in solid hydrogels was characterized with a modified Lowry assay. To illustrate the functional utility of this approach in a biological setting, we applied this system to develop materials that promote angiogenesis in an ex vivo aortic arch explant assay. We demonstrate the formation and invasion of new sprouts mediated by endothelial cells into the hydrogels from embedded embryonic chick aortic arches. Furthermore, we show that this capillary sprouting and three-dimensional migration of endothelial cells can be tuned by engineering the MMP-susceptibility of the hydrogels and the presence of functional immobilized adhesive ligands (CGRGDS vs. CGRGES peptide). The facile chemistry described and significant cellular responses observed suggest the usefulness of these materials in a variety of in vitro and ex vivo biologic investigations, and may aid in the design or refinement of material systems for a range of tissue engineering approaches.
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http://dx.doi.org/10.1016/j.biomaterials.2010.01.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2837100PMC
May 2010

Poly(ethylene glycol) hydrogels conjugated with a collagenase-sensitive fluorogenic substrate to visualize collagenase activity during three-dimensional cell migration.

Biomaterials 2007 Jul 14;28(20):3163-70. Epub 2007 Mar 14.

Department of Bioengineering, Rice University, P.O. Box 1892, MS 142, Houston, TX 77251-1892, USA.

We have developed collagenase-sensitive hydrogels by incorporating a collagenase-sensitive fluorogenic substrate (CS-FS) within the backbone of a polyethylene glycol (PEG) copolymer to visualize collagenase activity during three-dimensional cell migration. CS-FS was synthesized by conjugating Bodipy dyes to a peptide with collagenase-sensitive sequence, Leu-Gly-Pro-Ala (LGPA), and the products were grafted into the collagenase-sensitive PEG hydrogels. CS-FS both in solution and hydrogels had an increase in the fluorescence intensity after proteolytic degradation by collagenase, but not by non-targeted proteases nor in the absence of an enzyme. Fibroblasts inside the hydrogels conjugated with CS-FS spread and extended lamellipodia in three dimensions over several days, and their pericellular collagenase-mediated proteolysis of the hydrogel was visualized via confocal microscopy. A matrix metalloproteinase inhibitor, served as a negative control, significantly reduced the degradation rate of CS-FS by collagenase and prevented cell migration and cell-mediated collagenase activity inside these hydrogels. In summary, we have fabricated collagenase-sensitive hydrogels incorporated with CS-FS and successfully visualized the collagenase activity during three-dimensional cell migration.
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http://dx.doi.org/10.1016/j.biomaterials.2007.03.004DOI Listing
July 2007

Laser-scanning lithography (LSL) for the soft lithographic patterning of cell-adhesive self-assembled monolayers.

Biotechnol Bioeng 2006 Apr;93(6):1060-8

Department of Bioengineering, Rice University, P.O. Box 1892, MS-142, Houston, Texas 77251-1892, USA.

We report the development of laser-scanning lithography (LSL), which employs a laser-scanning confocal microscope to pattern photoresists that can be utilized, for example, in the fabrication of masters for use in soft lithography. This convenient technique provides even exposure across the entire view field and facilitates accurate alignment of successive photoresist exposures. Features on the scale of 3 microm have been achieved to date with a 10x objective (NA 0.45). Virtual masks, instructions for laser irradiation, were drawn using the Region of Interest (ROI) function of a Zeiss LSM 510 microscope. These regions were then exposed to a 458 nm argon laser for 32 micros (0.9 mW/microm(2)). Differential interference contrast (DIC) imaging was utilized with a non-destructive 514 nm argon laser as an immediate quality check of each exposure, to align successive exposures, and to reduce chromatic aberration between imaging and exposure. Developed masters were replica-molded with poly(dimethylsiloxane) (PDMS); these masters were then utilized for microcontact printing of cell-adhesive self-assembled monolayers (SAMs) to demonstrate the utility of this process. Initial studies confirmed that human dermal fibroblast adhesion and spreading were limited to cell-adhesive SAM areas. LSL is a rapid, flexible, and readily available technique that will accelerate master design and preparation; moreover, it can be applied to additional forms of photolithography and photopolymerization for studies in cell biology, biomaterials design and evaluation, materials science, and surface chemistry.
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http://dx.doi.org/10.1002/bit.20809DOI Listing
April 2006