191 research outputs found
Enhanced differentiation of retinal progenitor cells using microfabricated topographical cues
Due to the retinaās inability to replace photoreceptors lost during retinal degeneration, significant interest has been placed in methods to implant replacement cells. Polymer scaffolds are increasingly being studied as vehicles for cellular delivery to degenerated retinas. Previously, we fabricated poly(methyl methacrylate) thin film scaffolds that increased survival and integration of implanted retinal progenitor cells (RPCs). Additionally, these scaffolds minimized the trauma and cellular response associated with implantation of foreign bodies into mouse eyes. Here, we demonstrate that biodegradable polycaprolactone (PCL) thin film scaffolds can be fabricated with integrated microtopography. Microfabricated topography in a PCL thin film enhanced the attachment and organization of RPCs compared to unstructured surfaces. Using real-time quantitative polymerase chain reaction we also observed that attachment to microtopography induced cellular differentiation. RPCs grown on PCL thin films exhibited an increase in gene expression for the photoreceptor markers recoverin and rhodopsin, an increase in the glial and MĆ¼ller cell marker GFAP, and a decrease in SOX2 gene expression (a marker for undifferentiated progenitor cells) compared to cells grown on unmodified tissue culture polystyrene (TCPS)
Tunable Microfibers Suppress Fibrotic Encapsulation via Inhibition of TGFĪ² Signaling
Fibrotic encapsulation limits the efficacy and lifetime of implantable biomedical devices. Microtopography has shown promise in the regulation of myofibroblast differentiation, a key driver of fibrotic encapsulation. However, existing studies have not systematically isolated the requisite geometric parameters for suppression of myofibroblast differentiation via microtopography, and there has not been in vivo validation of this technology to date. To address these issues, a novel lamination method was developed to afford more control over topography dimensions. Specifically, in this study we focus on fiber length and its effect on myofibroblast differentiation. Fibroblasts cultured on films with microfibers exceeding 16 Ī¼m in length lost the characteristic morphology associated with myofibroblast differentiation, while shorter microfibers of 6 Ī¼m length failed to produce this phenotype. This increase in length corresponded to a 50% decrease in fiber stiffness, which acts as a mechanical cue to influence myofibroblast differentiation. Longer microfiber films suppressed expression of myofibroblast specific genes (Ī±SMA, Col1Ī±2, and Col3Ī±1) and TGFĪ² signaling components (TGFĪ²1 ligand, TGFĪ² receptor II, and Smad3). 16 Ī¼m long microfiber films implanted subcutaneously in a mouse wound-healing model generated a substantially thinner fibrotic capsule and less deposition of collagen in the wound bed. Together, these results identify a critical feature length threshold for microscale topography-mediated repression of fibrotic encapsulation. This study also demonstrates a simple and powerful strategy to improve surface biocompatibility and reduce fibrotic encapsulation around implanted materials
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Picoliter-volume inkjet printing into planar microdevice reservoirs for low-waste, high-capacity drug loading.
Oral delivery of therapeutics is the preferred route for systemic drug administration due to ease of access and improved patient compliance. However, many therapeutics suffer from low oral bioavailability due to low pH and enzymatic conditions, poor cellular permeability, and low residence time. Microfabrication techniques have been used to create planar, asymmetric microdevices for oral drug delivery to address these limitations. The geometry of these microdevices facilitates prolonged drug exposure with unidirectional release of drug toward gastrointestinal epithelium. While these devices have significantly enhanced drug permeability in vitro and in vivo, loading drug into the micron-scale reservoirs of the devices in a low-waste, high-capacity manner remains challenging. Here, we use picoliter-volume inkjet printing to load topotecan and insulin into planar microdevices efficiently. Following a simple surface functionalization step, drug solution can be spotted into the microdevice reservoir. We show that relatively high capacities of both topotecan and insulin can be loaded into microdevices in a rapid, automated process with little to no drug waste
Probing the luminal microenvironment of reconstituted epithelial microtissues.
Polymeric microparticles can serve as carriers or sensors to instruct or characterize tissue biology. However, incorporating microparticles into tissues for in vitro assays remains a challenge. We exploit three-dimensional cell-patterning technologies and directed epithelial self-organization to deliver microparticles to the lumen of reconstituted human intestinal microtissues. We also develop a novel pH-sensitive microsensor that can measure the luminal pH of reconstituted epithelial microtissues. These studies offer a novel approach for investigating luminal microenvironments and drug-delivery across epithelial barriers
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Changes in epithelial proportions and transcriptional state underlie major premenopausal breast cancer risks
The human breast undergoes lifelong remodeling in response to estrogen and progesterone, but hormone exposure also increases breast cancer risk. Here, we use single-cell analysis to identify distinct mechanisms through which breast composition and cell state affect hormone signaling. We show that prior pregnancy reduces the transcriptional response of hormone-responsive (HR+) epithelial cells, whereas high body mass index (BMI) reduces overall HR+ cell proportions. These distinct changes both impact neighboring cells by effectively reducing the magnitude of paracrine signals originating from HR+ cells. Because pregnancy and high BMI are known to protect against hormone-dependent breast cancer in premenopausal women, our findings directly link breast cancer risk with person-to-person heterogeneity in hormone responsiveness. More broadly, our findings illustrate how cell proportions and cell state can collectively impact cell communities through the action of cell-to-cell signaling networks
A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity
Developing tissues contain motile populations of cells that can self-organize into spatially ordered tissues based on differences in their interfacial surface energies. However, it is unclear how self-organization by this mechanism remains robust when interfacial energies become heterogeneous in either time or space. The ducts and acini of the human mammary gland are prototypical heterogeneous and dynamic tissues comprising two concentrically arranged cell types. To investigate the consequences of cellular heterogeneity and plasticity on cell positioning in the mammary gland, we reconstituted its self-organization from aggregates of primary cells in vitro. We find that self-organization is dominated by the interfacial energy of the tissueāECM boundary, rather than by differential homo- and heterotypic energies of cellācell interaction. Surprisingly, interactions with the tissueāECM boundary are binary, in that only one cell type interacts appreciably with the boundary. Using mathematical modeling and cell-type-specific knockdown of key regulators of cellācell cohesion, we show that this strategy of self-organization is robust to severe perturbations affecting cellācell contact formation. We also find that this mechanism of self-organization is conserved in the human prostate. Therefore, a binary interfacial interaction with the tissue boundary provides a flexible and generalizable strategy for forming and maintaining the structure of two-component tissues that exhibit abundant heterogeneity and plasticity. Our model also predicts that mutations affecting binary cellāECM interactions are catastrophic and could contribute to loss of tissue architecture in diseases such as breast cancer
Changes in epithelial proportions and transcriptional state underlie major premenopausal breast cancer risks
The human breast undergoes lifelong remodeling in response to estrogen and progesterone, but hormone exposure also increases breast cancer risk. Here, we use single-cell analysis to identify distinct mechanisms through which breast composition and cell state affect hormone signaling. We show that prior pregnancy reduces the transcriptional response of hormone-responsive (HR+) epithelial cells, whereas high body mass index (BMI) reduces overall HR+ cell proportions. These distinct changes both impact neighboring cells by effectively reducing the magnitude of paracrine signals originating from HR+ cells. Because pregnancy and high BMI are known to protect against hormone-dependent breast cancer in premenopausal women, our findings directly link breast cancer risk with person-to-person heterogeneity in hormone responsiveness. More broadly, our findings illustrate how cell proportions and cell state can collectively impact cell communities through the action of cell-to-cell signaling networks
Microdomain heterogeneity in 3D affects the mechanics of neonatal cardiac myocyte contraction
Abstract Cardiac muscle cells are known to adapt to their physical surroundings, optimizing intracellular organization and contractile function for a given culture environment. A previously developed in vitro model system has shown that the inclusion of discrete microscale domains (or microrods) in three dimensions (3D) can alter long-term growth responses of neonatal ventricular myocytes. The aim of this work was to understand how cellular contact with such a domain affects various mechanical changes involved in cardiac muscle cell remodeling. Myocytes were maintained in 3D gels over 5 days in the presence or absence of 100 ā Āµm-long microrods, and the effect of this local heterogeneity on cell behavior was analyzed via several imaging techniques. Microrod abutment resulted in approximately twofold increases in the maximum displacement of spontaneously beating myocytes, as based on confocal microscopy scans of the gel xy-plane or the myocyte long axis. In addition, microrods caused significant increases in the Electronic supplementary material The online version of this articl
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Local injections of Ī²-NGF accelerates endochondral fracture repair by promoting cartilage to bone conversion.
There are currently no pharmacological approaches in fracture healing designed to therapeutically stimulate endochondral ossification. In this study, we test nerve growth factor (NGF) as an understudied therapeutic for fracture repair. We first characterized endogenous expression of Ngf and its receptor tropomyosin receptor kinase A (TrkA) during tibial fracture repair, finding that they peak during the cartilaginous phase. We then tested two injection regimens and found that local Ī²-NGF injections during the endochondral/cartilaginous phase promoted osteogenic marker expression. Gene expression data from Ī²-NGF stimulated cartilage callus explants show a promotion in markers associated with endochondral ossification such as Ihh, Alpl, and Sdf-1. Gene ontology enrichment analysis revealed the promotion of genes associated with Wnt activation, PDGF- and integrin-binding. Subsequent histological analysis confirmed Wnt activation following local Ī²-NGF injections. Finally, we demonstrate functional improvements to bone healing following local Ī²-NGF injections which resulted in a decrease in cartilage and increase of bone volume. Moreover, the newly formed bone contained higher trabecular number, connective density, and bone mineral density. Collectively, we demonstrate Ī²-NGF\u27s ability to promote endochondral repair in a murine model and uncover mechanisms that will serve to further understand the molecular switches that occur during cartilage to bone transformation
Fabrication of Sealed Nanostraw Microdevices for Oral Drug Delivery
The oral route is preferred for systemic drug administration and provides direct access to diseased tissue of the gastrointestinal (GI) tract. However, many drugs have poor absorption upon oral administration due to damaging enzymatic and pH conditions, mucus and cellular permeation barriers, and limited time for drug dissolution. To overcome these limitations and enhance oral drug absorption, micron-scale devices with planar, asymmetric geometries, termed microdevices, have been designed to adhere to the lining of the GI tract and release drug at high concentrations directly toward GI epithelium. Here we seal microdevices with nanostraw membranesāporous nanostructured biomolecule delivery substratesāto enhance the properties of these devices. We demonstrate that the nanostraws facilitate facile drug loading and tunable drug release, limit the influx of external molecules into the sealed drug reservoir, and increase the adhesion of devices to epithelial tissue. These findings highlight the potential of nanostraw microdevices to enhance the oral absorption of a wide range of therapeutics by binding to the lining of the GI tract, providing prolonged and proximal drug release, and reducing the exposure of their payload to drug-degrading biomolecules
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