The Experts below are selected from a list of 1953 Experts worldwide ranked by ideXlab platform
David L Kaplan - One of the best experts on this subject based on the ideXlab platform.
-
Self-Folding 3D Silk Biomaterial Rolls to Facilitate Axon and Bone Regeneration.
Advanced healthcare materials, 2020Co-Authors: Yimin Huang, Chiara E Ghezzi, David L Kaplan, Vincent Fitzpatrick, Nan Zheng, Ran Cheng, Heyu Huang, Chen YangAbstract:Biomaterial scaffold designs are needed for self-organizing features related to tissue formation while also simplifying the fabrication processes involved. Toward this goal, Silk protein-based self-folding scaffolds to support 3D cell culture, while providing directional guidance and promotion of cell growth and differentiation, are reported. A simple and robust one-step self-folding approach is developed using bilayers consisting of a hydrogel and Silk Film in aqueous solution. The 3D Silk rolls, with patterns transferred from the initially prepared 2D Films, guide the directional outgrowth of neurites and also promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The osteogenic outcomes are further supported by enhanced biomechanical performance. By utilizing this self-folding method, cocultures of neurons and hMSCs are achieved by patterning cells on Silk Films and then converting these materials into a 3D format with rolling, mimicking aspects of the structure of osteons and providing physiologically relevant structures to promote bone regeneration. These results demonstrate the utility of self-folded Silk rolls as efficient scaffold systems for tissue regeneration, while exploiting relatively simple 2D designs programmed to form more complex 3D structures.
-
functional rf trilayer sensors for tooth mounted wireless monitoring of the oral cavity and food consumption
Advanced Materials, 2018Co-Authors: Peter Tseng, Bradley Napier, Logan P Garbarini, David L KaplanAbstract:Wearable devices have emerged as powerful tools for personalized healthcare in spite of some challenges that limit their widespread applicability as continuous monitors of physiological information. Here, a materials-based strategy to add utility to traditional dielectric sensors by developing a conformal radiofrequency (RF) construct composed of an active layer encapsulated between two reverse-facing split ring resonators is applied. These small (down to 2 mm × 2 mm) passive dielectric sensors possess enhanced sensitivity and can be further augmented by functionalization of this interlayer material. Demonstrator devices are shown where the interlayer is: (i) a porous Silk Film, and (ii) a modified PNIPAM hydrogel that swells with pH or temperature. In vivo use is demonstrated by adhesion of the device on tooth enamel to detect foods during human ingestion. Such sensors can be easily multiplexed and yield data-rich temporal information during the diffusion of analytes within the trilayer structure. This format could be extended to a suite of interlayer materials for sensing devices of added use and specificity.
-
multi layered Silk Film coculture system for human corneal epithelial and stromal stem cells
Journal of Tissue Engineering and Regenerative Medicine, 2018Co-Authors: Emily A Gosselin, Chiara E Ghezzi, James L. Funderburgh, Tess Torregrosa, Alexandra C Mendelsohn, Rachel Gomes, David L KaplanAbstract:With insufficient options to meet the clinical demand for cornea transplants, one emerging area of emphasis is on cornea tissue engineering. In the present study, the goal was to combine the corneal stroma and epithelium into one co-culture system, in order to monitor both human corneal stromal stem cell (hCSSC) and human corneal epithelial cell (hCE) growth and differentiation into keratocytes and differentiated epithelium in these 3D tissue systems in vitro. Co-culture conditions were first optimized, including the medium, air/liquid interface culture, and surface topography and chemistry of biomaterial scaffold Films based on Silk protein. The Silk was used as scaffolding for both stromal and epithelial tissue layers because it is cell compatible, can be surface patterned, and is optically clear. Next, the effects of proliferating and differentiating hCEs and hCSSCs in this in vitro system were studied, including the effects on cell proliferation, matrix formation by immunochemistry, and gene expression by RT-qPCR. The incorporation of both cell types into the co-culture system demonstrated more complete differentiation and growth for both cell types compared to the corneal stromal cells and corneal epithelial cells alone. Silk Films for corneal epithelial culture were optimized to combine a 4.0 micron-scale surface pattern with bulk-loaded collagen type IV. Differentiation of each cell type was in evidence based on increased expression of corneal stroma and epithelial proteins and transcript levels after 6 weeks in co-culture on the optimized Silk scaffolds. This article is protected by copyright. All rights reserved.
-
Multi-layered Silk Film coculture system for human corneal epithelial and stromal stem cells.
Journal of tissue engineering and regenerative medicine, 2017Co-Authors: Emily A Gosselin, Chiara E Ghezzi, James L. Funderburgh, Tess Torregrosa, Alexandra C Mendelsohn, Rachel Gomes, David L KaplanAbstract:With insufficient options to meet the clinical demand for cornea transplants, one emerging area of emphasis is on cornea tissue engineering. In the present study, the goal was to combine the corneal stroma and epithelium into one coculture system, to monitor both human corneal stromal stem cell (hCSSC) and human corneal epithelial cell (hCE) growth and differentiation into keratocytes and differentiated epithelium in these three-dimensional tissue systems in vitro. Coculture conditions were first optimized, including the medium, air-liquid interface culture, and surface topography and chemistry of biomaterial scaffold Films based on Silk protein. The Silk was used as scaffolding for both stromal and epithelial tissue layers because it is cell compatible, can be surface patterned, and is optically clear. Next, the effects of proliferating and differentiating hCEs and hCSSCs were studied in this in vitro system, including the effects on cell proliferation, matrix formation by immunochemistry, and gene expression by quantitative reverse transcription-polymerase chain reaction. The incorporation of both cell types into the coculture system demonstrated more complete differentiation and growth for both cell types compared to the corneal stromal cells and corneal epithelial cells alone. Silk Films for corneal epithelial culture were optimized to combine a 4.0-μm-scale surface pattern with bulk-loaded collagen type IV. Differentiation of each cell type was in evidence based on increased expression of corneal stroma and epithelial proteins and transcript levels after 6 weeks in coculture on the optimized Silk scaffolds.
-
3D Functional Corneal Stromal Tissue Equivalent Based on Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture
PLOS ONE, 2017Co-Authors: Chiara E Ghezzi, Benedetto Marelli, James L. Funderburgh, David L KaplanAbstract:The worldwide need for human cornea equivalents continues to grow. Few clinical options are limited to allogenic and synthetic material replacements. We hypothesized that tissue engineered human cornea systems based on mechanically robust, patterned, porous, thin, optically clear Silk protein Films, in combination with human corneal stromal stem cells (hCSSCs), would generate 3D functional corneal stroma tissue equivalents, in comparison to previously developed 2D approaches. Silk Film contact guidance was used to control the alignment and distribution of hCSSCs on RGD-treated single porous Silk Films, which were then stacked in an orthogonally, multi-layered architecture and cultured for 9 weeks. These systems were compared similar systems generated with human corneal fibroblasts (hCFs). Both cell types were viable and preferentially aligned along the biomaterial patterns for up to 9 weeks in culture. H&E histological sections showed that the systems seeded with the hCSSCs displayed ECM production throughout the entire thickness of the constructs. In addition, the ECM proteins tested positive for keratocyte-specific tissue markers, including keratan sulfate, lumican, and keratocan. The quantification of hCSSC gene expression of keratocyte-tissue markers, including keratocan, lumican, human aldehyde dehydrogenase 3A1 (ALDH3A1), prostaglandin D2 synthase (PTDGS), and pyruvate dehydrogenase kinase, isozyme 4 (PDK4), within the 3D tissue systems demonstrated upregulation when compared to 2D single Silk Films and to the systems generated with the hCFs. Furthermore, the production of ECM from the hCSSC seeded systems and subsequent remodeling of the initial matrix significantly improved cohesiveness and mechanical performance of the constructs, while maintaining transparency after 9 weeks.
Chiara E Ghezzi - One of the best experts on this subject based on the ideXlab platform.
-
Self-Folding 3D Silk Biomaterial Rolls to Facilitate Axon and Bone Regeneration.
Advanced healthcare materials, 2020Co-Authors: Yimin Huang, Chiara E Ghezzi, David L Kaplan, Vincent Fitzpatrick, Nan Zheng, Ran Cheng, Heyu Huang, Chen YangAbstract:Biomaterial scaffold designs are needed for self-organizing features related to tissue formation while also simplifying the fabrication processes involved. Toward this goal, Silk protein-based self-folding scaffolds to support 3D cell culture, while providing directional guidance and promotion of cell growth and differentiation, are reported. A simple and robust one-step self-folding approach is developed using bilayers consisting of a hydrogel and Silk Film in aqueous solution. The 3D Silk rolls, with patterns transferred from the initially prepared 2D Films, guide the directional outgrowth of neurites and also promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The osteogenic outcomes are further supported by enhanced biomechanical performance. By utilizing this self-folding method, cocultures of neurons and hMSCs are achieved by patterning cells on Silk Films and then converting these materials into a 3D format with rolling, mimicking aspects of the structure of osteons and providing physiologically relevant structures to promote bone regeneration. These results demonstrate the utility of self-folded Silk rolls as efficient scaffold systems for tissue regeneration, while exploiting relatively simple 2D designs programmed to form more complex 3D structures.
-
multi layered Silk Film coculture system for human corneal epithelial and stromal stem cells
Journal of Tissue Engineering and Regenerative Medicine, 2018Co-Authors: Emily A Gosselin, Chiara E Ghezzi, James L. Funderburgh, Tess Torregrosa, Alexandra C Mendelsohn, Rachel Gomes, David L KaplanAbstract:With insufficient options to meet the clinical demand for cornea transplants, one emerging area of emphasis is on cornea tissue engineering. In the present study, the goal was to combine the corneal stroma and epithelium into one co-culture system, in order to monitor both human corneal stromal stem cell (hCSSC) and human corneal epithelial cell (hCE) growth and differentiation into keratocytes and differentiated epithelium in these 3D tissue systems in vitro. Co-culture conditions were first optimized, including the medium, air/liquid interface culture, and surface topography and chemistry of biomaterial scaffold Films based on Silk protein. The Silk was used as scaffolding for both stromal and epithelial tissue layers because it is cell compatible, can be surface patterned, and is optically clear. Next, the effects of proliferating and differentiating hCEs and hCSSCs in this in vitro system were studied, including the effects on cell proliferation, matrix formation by immunochemistry, and gene expression by RT-qPCR. The incorporation of both cell types into the co-culture system demonstrated more complete differentiation and growth for both cell types compared to the corneal stromal cells and corneal epithelial cells alone. Silk Films for corneal epithelial culture were optimized to combine a 4.0 micron-scale surface pattern with bulk-loaded collagen type IV. Differentiation of each cell type was in evidence based on increased expression of corneal stroma and epithelial proteins and transcript levels after 6 weeks in co-culture on the optimized Silk scaffolds. This article is protected by copyright. All rights reserved.
-
Multi-layered Silk Film coculture system for human corneal epithelial and stromal stem cells.
Journal of tissue engineering and regenerative medicine, 2017Co-Authors: Emily A Gosselin, Chiara E Ghezzi, James L. Funderburgh, Tess Torregrosa, Alexandra C Mendelsohn, Rachel Gomes, David L KaplanAbstract:With insufficient options to meet the clinical demand for cornea transplants, one emerging area of emphasis is on cornea tissue engineering. In the present study, the goal was to combine the corneal stroma and epithelium into one coculture system, to monitor both human corneal stromal stem cell (hCSSC) and human corneal epithelial cell (hCE) growth and differentiation into keratocytes and differentiated epithelium in these three-dimensional tissue systems in vitro. Coculture conditions were first optimized, including the medium, air-liquid interface culture, and surface topography and chemistry of biomaterial scaffold Films based on Silk protein. The Silk was used as scaffolding for both stromal and epithelial tissue layers because it is cell compatible, can be surface patterned, and is optically clear. Next, the effects of proliferating and differentiating hCEs and hCSSCs were studied in this in vitro system, including the effects on cell proliferation, matrix formation by immunochemistry, and gene expression by quantitative reverse transcription-polymerase chain reaction. The incorporation of both cell types into the coculture system demonstrated more complete differentiation and growth for both cell types compared to the corneal stromal cells and corneal epithelial cells alone. Silk Films for corneal epithelial culture were optimized to combine a 4.0-μm-scale surface pattern with bulk-loaded collagen type IV. Differentiation of each cell type was in evidence based on increased expression of corneal stroma and epithelial proteins and transcript levels after 6 weeks in coculture on the optimized Silk scaffolds.
-
3D Functional Corneal Stromal Tissue Equivalent Based on Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture
PLOS ONE, 2017Co-Authors: Chiara E Ghezzi, Benedetto Marelli, James L. Funderburgh, David L KaplanAbstract:The worldwide need for human cornea equivalents continues to grow. Few clinical options are limited to allogenic and synthetic material replacements. We hypothesized that tissue engineered human cornea systems based on mechanically robust, patterned, porous, thin, optically clear Silk protein Films, in combination with human corneal stromal stem cells (hCSSCs), would generate 3D functional corneal stroma tissue equivalents, in comparison to previously developed 2D approaches. Silk Film contact guidance was used to control the alignment and distribution of hCSSCs on RGD-treated single porous Silk Films, which were then stacked in an orthogonally, multi-layered architecture and cultured for 9 weeks. These systems were compared similar systems generated with human corneal fibroblasts (hCFs). Both cell types were viable and preferentially aligned along the biomaterial patterns for up to 9 weeks in culture. H&E histological sections showed that the systems seeded with the hCSSCs displayed ECM production throughout the entire thickness of the constructs. In addition, the ECM proteins tested positive for keratocyte-specific tissue markers, including keratan sulfate, lumican, and keratocan. The quantification of hCSSC gene expression of keratocyte-tissue markers, including keratocan, lumican, human aldehyde dehydrogenase 3A1 (ALDH3A1), prostaglandin D2 synthase (PTDGS), and pyruvate dehydrogenase kinase, isozyme 4 (PDK4), within the 3D tissue systems demonstrated upregulation when compared to 2D single Silk Films and to the systems generated with the hCFs. Furthermore, the production of ECM from the hCSSC seeded systems and subsequent remodeling of the initial matrix significantly improved cohesiveness and mechanical performance of the constructs, while maintaining transparency after 9 weeks.
-
3D Functional Corneal Stromal Tissue Equivalent Based on Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture
PLOS ONE, 2017Co-Authors: Chiara E Ghezzi, Benedetto Marelli, James L. Funderburgh, Fiorenzo G Omenetto, David L KaplanAbstract:The worldwide need for human cornea equivalents continues to grow. Few clinical options are limited to allogenic and synthetic material replacements. We hypothesized that tissue engineered human cornea systems based on mechanically robust, patterned, porous, thin, optically clear Silk protein Films, in combination with human corneal stromal stem cells (hCSSCs), would generate 3D functional corneal stroma tissue equivalents, in comparison to previously developed 2D approaches. Silk Film contact guidance was used to control the alignment and distribution of hCSSCs on RGD-treated single porous Silk Films, which were then stacked in an orthogonally, multi-layered architecture and cultured for 9 weeks. These systems were compared similar systems generated with human corneal fibroblasts (hCFs). Both cell types were viable and preferentially aligned along the biomaterial patterns for up to 9 weeks in culture. H&E histological sections showed that the systems seeded with the hCSSCs displayed ECM production throughout the entire thickness of the constructs. In addition, the ECM proteins tested positive for keratocyte-specific tissue markers, including keratan sulfate, lumican, and keratocan. The quantification of hCSSC gene expression of keratocyte-tissue markers, including keratocan, lumican, human aldehyde dehydrogenase 3A1 (ALDH3A1), prostaglandin D2 synthase (PTDGS), and pyruvate dehydrogenase kinase, isozyme 4 (PDK4), within the 3D tissue systems demonstrated upregulation when compared to 2D single Silk Films and to the systems generated with the hCFs. Furthermore, the production of ECM from the hCSSC seeded systems and subsequent remodeling of the initial matrix significantly improved cohesiveness and mechanical performance of the constructs, while maintaining transparency after 9 weeks.
Mark I. Rosenblatt - One of the best experts on this subject based on the ideXlab platform.
-
Silk Films with nanotopography and extracellular proteins enhance corneal epithelial wound healing.
Scientific reports, 2021Co-Authors: Yuncin Luo, Kai B. Kang, Victor H. Guaiquil, Qiang Zhou, Rachel Sartaj, Michael G Sun, Mark I. RosenblattAbstract:Corneal wound healing depends on extracellular matrix (ECM) and topographical cues that modulate migration and proliferation of regenerating cells. In our study, Silk Films with either flat or nanotopography patterned parallel ridge widths of 2000, 1000, 800 nm surfaces were combined with ECMs which include collagen type I (collagen I), fibronectin, laminin, and Poly-D-Lysine to accelerate corneal wound healing. Silk Films with 800 nm ridge width provided better cell spreading and wound recovery than other size topographies. Coating 800 nm patterned Silk Films with collagen I proves to optimally further increased mouse and rabbit corneal epithelial cells growth and wound recovery. This enhanced cellular response correlated with redistribution and increase in size and total amount of focal adhesion. Transcriptomics and signaling pathway analysis suggested that Silk topography regulates cell behaviors via actin nucleation ARP-WASP complex pathway, which regulate filopodia formation. This mechanism was further explored and inhibition of Cdc42, a key protein in this pathway, delayed wound healing and decreased the length, density, and alignment of filopodia. Inhibition of Cdc42 in vivo resulted in delayed re-epithelization of injured corneas. We conclude that Silk Film nanotopography in combination with collagen I constitutes a better substrate for corneal wound repair than either nanotopography or ECM alone.
-
The Effect of Micro- and Nanoscale Surface Topographies on Silk on Human Corneal Limbal Epithelial Cell Differentiation.
Scientific reports, 2019Co-Authors: Kai B. Kang, Brian Lawrence, Aihong Liu, X. Raymond Gao, Victor H. Guaiquil, Mark I. RosenblattAbstract:We previously reported that micro- and nano-scale topographic pitch created on Silk Films mimic features of the corneal basement membrane by providing biophysical cues to direct corneal epithelial cell adherence and migration. However, the effect of these topographical features on corneal limbal epithelial cell differentiation has not been explored. We hypothesize in the current study that various topographical pitch created on Silk may affect corneal epithelial stem cell differentiation and alter the expression of genes involved in cell differentiation and self-renewal. We patterned Silk Films with different topographic pitch via soft lithography and observed human corneal limbal epithelial cell behavior. Colony forming assay demonstrated increased colony forming efficiency on patterned Silk Films. Cells cultured on nanoscale patterned Silk Films also expressed lower levels of putative keratocyte differentiation markers and higher levels of putative limbal stem cell markers. RNA-Seq analysis further implicated the involvement of pathways related to stem cell differentiation and self-renewal, including Notch, ERK/MAPK and Wnt/β-catenin signaling. We conclude that patterned Silk Film substrates can be used as scaffolds and provide biophysical cues to corneal limbal stem cells that may maintain corneal epithelial stem cells at a less differentiated state.
-
Micro- and Nanoscale Topographies on Silk Regulate Gene Expression of Human Corneal Epithelial Cells
Investigative ophthalmology & visual science, 2017Co-Authors: Kai B. Kang, Brian Lawrence, Aihong Liu, X. Raymond Gao, Victor H. Guaiquil, Yuncin Luo, Qiang Zhou, Mark I. RosenblattAbstract:Purpose Corneal basement membrane has topographical features that provide biophysical cues to direct cell adherence, migration, and proliferation. In this study, we hypothesize that varying topographic pitch created on Silk Films can alter epithelial cell morphology, adhesion, and the genetic expression involved in cytoskeletal dynamics-related pathways. Methods Silicon wafers with parallel ridge widths of 2000, 1000, and 800 nm were produced and used to pattern Silk Films via soft lithography. Human corneal epithelial cells were cultured onto Silk. After 72 hours of incubation, images were taken to study cell morphology and alignment. Cytoskeletal structures were studied by immunofluorescent staining. RNA was collected from cultured cells to perform RNA-Seq transcriptome analysis using the Illumina Hiseq 2500 sequencing system. Differentially expressed genes were identified using DNAstar Qseq then verified using quantitative real-time PCR. These genes were used to perform pathway analyses using Ingenuity Pathways Analysis. Results Primary human corneal epithelial cell alignment to the surface pattern was the greatest on 1000-nm features. Fluorescent microscopy of f-actin staining showed cell cytoskeleton alignment either in parallel (2000 nm) or perpendicular (1000 and 800 nm) to the long feature axis. Z-stack projection of vinculin staining indicated increased focal adhesion formation localized on the cellular basal surface. RNA-seq analysis revealed differentially expressed genes involved in actin organization, integrin signaling, and focal adhesion kinase signaling (-log (P)>5). Conclusions Patterned Silk Film substrates may serve as a scaffold and provide biophysical cues to corneal epithelial cells that change their gene expression, alter cellular adherence, morphology, and may offer a promising customizable material for use in ocular surface repair.
-
Silk Film Topography Directs Collective Epithelial Cell Migration
PloS one, 2012Co-Authors: Brian Lawrence, Zhi Pan, Mark I. RosenblattAbstract:The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned Silk Film surfaces. The Silk Film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization.
-
Human corneal limbal epithelial cell response to varying Silk Film geometric topography in vitro.
Acta biomaterialia, 2012Co-Authors: Brian Lawrence, David L Kaplan, Zhi Pan, Aihong Liu, Mark I. RosenblattAbstract:Silk fibroin Films are a promising class of biomaterials that have a number of advantages for use in ophthalmic applications due to their transparent nature, mechanical properties and minimal inflammatory response upon implantation. Freestanding Silk Films with parallel line and concentric ring topographies were generated for in vitro characterization of human corneal limbal epithelial (HCLE) cell response upon differing geometric patterned surfaces. Results indicated that Silk Film topography significantly affected initial HCLE culture substrate attachment, cellular alignment, cell-to-cell contact formation, actin cytoskeleton alignment and focal adhesion (FA) localization. Most notably, parallel line patterned surfaces displayed a 36-54% increase on average in initial cell attachment, which corresponded to a more than 2-fold increase in FA localization when compared to other Silk Film surfaces and controls. In addition, distinct localization of FA formation was observed along the edges for all patterned Silk Film topographies. In conclusion, Silk Film feature topography appears to help direct corneal epithelial cell response and cytoskeleton development, especially with regard to FA distribution, in vitro.
Fiorenzo G Omenetto - One of the best experts on this subject based on the ideXlab platform.
-
3D Functional Corneal Stromal Tissue Equivalent Based on Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture
PLOS ONE, 2017Co-Authors: Chiara E Ghezzi, Benedetto Marelli, James L. Funderburgh, Fiorenzo G Omenetto, David L KaplanAbstract:The worldwide need for human cornea equivalents continues to grow. Few clinical options are limited to allogenic and synthetic material replacements. We hypothesized that tissue engineered human cornea systems based on mechanically robust, patterned, porous, thin, optically clear Silk protein Films, in combination with human corneal stromal stem cells (hCSSCs), would generate 3D functional corneal stroma tissue equivalents, in comparison to previously developed 2D approaches. Silk Film contact guidance was used to control the alignment and distribution of hCSSCs on RGD-treated single porous Silk Films, which were then stacked in an orthogonally, multi-layered architecture and cultured for 9 weeks. These systems were compared similar systems generated with human corneal fibroblasts (hCFs). Both cell types were viable and preferentially aligned along the biomaterial patterns for up to 9 weeks in culture. H&E histological sections showed that the systems seeded with the hCSSCs displayed ECM production throughout the entire thickness of the constructs. In addition, the ECM proteins tested positive for keratocyte-specific tissue markers, including keratan sulfate, lumican, and keratocan. The quantification of hCSSC gene expression of keratocyte-tissue markers, including keratocan, lumican, human aldehyde dehydrogenase 3A1 (ALDH3A1), prostaglandin D2 synthase (PTDGS), and pyruvate dehydrogenase kinase, isozyme 4 (PDK4), within the 3D tissue systems demonstrated upregulation when compared to 2D single Silk Films and to the systems generated with the hCFs. Furthermore, the production of ECM from the hCSSC seeded systems and subsequent remodeling of the initial matrix significantly improved cohesiveness and mechanical performance of the constructs, while maintaining transparency after 9 weeks.
-
all water based electron beam lithography using Silk as a resist
Nature Nanotechnology, 2014Co-Authors: Sunghwan Kim, Benedetto Marelli, David L Kaplan, Eun Seok Gil, Hu Tao, Mark A. Brenckle, Alexander N Mitropoulos, Konstantinos Tsioris, Fiorenzo G OmenettoAbstract:Traditional nanofabrication techniques often require complex lithographic steps and the use of toxic chemicals. To move from the laboratory scale to large scales, nanofabrication should be carried out using alternative procedures that are simple, inexpensive and use non-toxic solvents. Recent efforts have focused on nanoimprinting and the use of organic resists (such as quantum dot-polymer hybrids, DNA and poly(ethylene glycol)), which still require, for the most part, noxious chemicals for processing. Significant advances have been achieved using 'green' resists that can be developed with water, but so far these approaches have suffered from low electron sensitivity, line edge roughness and scalability constraints. Here, we present the use of Silk as a natural and biofunctional resist for electron-beam lithography. The process is entirely water-based, starting with the Silk aqueous solution and ending with simple development of the exposed Silk Film in water. Because of its polymorphic crystalline structure, Silk can be used either as a positive or negative resist through interactions with an electron beam. Moreover, Silk can be easily modified, thereby enabling a variety of 'functional resists', including biologically active versions. As a proof of principle of the viability of all-water-based Silk electron-beam lithography (EBL), we fabricate nanoscale photonic lattices using both neat Silk and Silk doped with quantum dots, green fluorescent proteins (GFPs) or horseradish peroxidase (HRP).
-
Rapid nano impact printing of Silk biopolymer thin Films
Journal of Micromechanics and Microengineering, 2011Co-Authors: Robert D. White, David L Kaplan, Caprice Gray, Ethan Mandelup, Jason J. Amsden, Fiorenzo G OmenettoAbstract:In this paper, nano impact printing of Silk biopolymer Films is described. An indenter is rapidly accelerated and transfers the nanopattern from a silicon master into the Silk Film during an impact event that occurs in less than 1 ms. Contact stresses of greater than 100 MPa can be achieved during the short impact period with low power and inexpensive hardware. Ring shaped features with a diameter of 2 µm and a ring width of 100–200 nm were successfully transferred into untreated Silk Films using this method at room temperature. Mechanical modeling was carried out to determine the contact stress distribution, and demonstrates that imprinting can occur for contact stresses of less than 2 MPa. Thermal characterization at the impact location shows that raising the temperature to 70 °C has only a limited effect on pattern transfer. Contact stresses of greater than approximately 100 MPa result in excessive deformation of the Film and poor pattern transfer.
-
Gold nanoparticle-doped biocompatible Silk Films as a path to implantable thermo-electrically wireless powering devices
Applied Physics Letters, 2010Co-Authors: Hu Tao, David L Kaplan, Sean M. Siebert, Mark A. Brenckle, Richard D. Averitt, Mark Cronin-golomb, Fiorenzo G OmenettoAbstract:In this paper, we report on gold nanoparticle (GNP) doped Silk Films as an implantable and degradable heating element activated by light, which can be potentially used for wireless powering of implanted microdevices. Proof-of-concept experiments have been conducted by casting a GNP doped Silk Film on a miniature thermal-power chip, which generates ∼20 mW when illuminated by a green laser with an output power of 450 mW/mm2 at 532 nm.
-
effect of hydration on Silk Film material properties
Macromolecular Bioscience, 2010Co-Authors: Fiorenzo G Omenetto, Scott E. Wharram, Jonathan A. Kluge, Gary G. Leisk, Ia Lawrence, Mark I Rosenbla, David L KaplaAbstract:Effects of hydration on Silk fibroin Film material properties were investigated for water-annealed and MeOH treated samples. After hydration, thickness increased 60% for MeOH immersed Films, while water-annealed samples remained constant. TGA determined MeOH immersed Films had an 80% mass loss due to water, while water-annealed had a 40% mass loss. O2 permeability was greater in MeOH immersed Films with Dk values of 10 (10 −11 ·mLO2·cm)/(cm·s·mmHg), while water-annealed Films had Dk values of 2 (10 −11 ·mLO2·cm)/(cm·s·mmHg). All Films showed a decrease in Young’s modulus and increased plastic deformation by two orders of magnitude when submerged in saline solution. FTIR revealed water-annealed Films increased in β-sheet content with increasing water vapor, while MeOH immersed Films did not change.
Brian Lawrence - One of the best experts on this subject based on the ideXlab platform.
-
The Effect of Micro- and Nanoscale Surface Topographies on Silk on Human Corneal Limbal Epithelial Cell Differentiation.
Scientific reports, 2019Co-Authors: Kai B. Kang, Brian Lawrence, Aihong Liu, X. Raymond Gao, Victor H. Guaiquil, Mark I. RosenblattAbstract:We previously reported that micro- and nano-scale topographic pitch created on Silk Films mimic features of the corneal basement membrane by providing biophysical cues to direct corneal epithelial cell adherence and migration. However, the effect of these topographical features on corneal limbal epithelial cell differentiation has not been explored. We hypothesize in the current study that various topographical pitch created on Silk may affect corneal epithelial stem cell differentiation and alter the expression of genes involved in cell differentiation and self-renewal. We patterned Silk Films with different topographic pitch via soft lithography and observed human corneal limbal epithelial cell behavior. Colony forming assay demonstrated increased colony forming efficiency on patterned Silk Films. Cells cultured on nanoscale patterned Silk Films also expressed lower levels of putative keratocyte differentiation markers and higher levels of putative limbal stem cell markers. RNA-Seq analysis further implicated the involvement of pathways related to stem cell differentiation and self-renewal, including Notch, ERK/MAPK and Wnt/β-catenin signaling. We conclude that patterned Silk Film substrates can be used as scaffolds and provide biophysical cues to corneal limbal stem cells that may maintain corneal epithelial stem cells at a less differentiated state.
-
Micro- and Nanoscale Topographies on Silk Regulate Gene Expression of Human Corneal Epithelial Cells
Investigative ophthalmology & visual science, 2017Co-Authors: Kai B. Kang, Brian Lawrence, Aihong Liu, X. Raymond Gao, Victor H. Guaiquil, Yuncin Luo, Qiang Zhou, Mark I. RosenblattAbstract:Purpose Corneal basement membrane has topographical features that provide biophysical cues to direct cell adherence, migration, and proliferation. In this study, we hypothesize that varying topographic pitch created on Silk Films can alter epithelial cell morphology, adhesion, and the genetic expression involved in cytoskeletal dynamics-related pathways. Methods Silicon wafers with parallel ridge widths of 2000, 1000, and 800 nm were produced and used to pattern Silk Films via soft lithography. Human corneal epithelial cells were cultured onto Silk. After 72 hours of incubation, images were taken to study cell morphology and alignment. Cytoskeletal structures were studied by immunofluorescent staining. RNA was collected from cultured cells to perform RNA-Seq transcriptome analysis using the Illumina Hiseq 2500 sequencing system. Differentially expressed genes were identified using DNAstar Qseq then verified using quantitative real-time PCR. These genes were used to perform pathway analyses using Ingenuity Pathways Analysis. Results Primary human corneal epithelial cell alignment to the surface pattern was the greatest on 1000-nm features. Fluorescent microscopy of f-actin staining showed cell cytoskeleton alignment either in parallel (2000 nm) or perpendicular (1000 and 800 nm) to the long feature axis. Z-stack projection of vinculin staining indicated increased focal adhesion formation localized on the cellular basal surface. RNA-seq analysis revealed differentially expressed genes involved in actin organization, integrin signaling, and focal adhesion kinase signaling (-log (P)>5). Conclusions Patterned Silk Film substrates may serve as a scaffold and provide biophysical cues to corneal epithelial cells that change their gene expression, alter cellular adherence, morphology, and may offer a promising customizable material for use in ocular surface repair.
-
Silk Film Topography Directs Collective Epithelial Cell Migration
PloS one, 2012Co-Authors: Brian Lawrence, Zhi Pan, Mark I. RosenblattAbstract:The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned Silk Film surfaces. The Silk Film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization.
-
Human corneal limbal epithelial cell response to varying Silk Film geometric topography in vitro.
Acta biomaterialia, 2012Co-Authors: Brian Lawrence, David L Kaplan, Zhi Pan, Aihong Liu, Mark I. RosenblattAbstract:Silk fibroin Films are a promising class of biomaterials that have a number of advantages for use in ophthalmic applications due to their transparent nature, mechanical properties and minimal inflammatory response upon implantation. Freestanding Silk Films with parallel line and concentric ring topographies were generated for in vitro characterization of human corneal limbal epithelial (HCLE) cell response upon differing geometric patterned surfaces. Results indicated that Silk Film topography significantly affected initial HCLE culture substrate attachment, cellular alignment, cell-to-cell contact formation, actin cytoskeleton alignment and focal adhesion (FA) localization. Most notably, parallel line patterned surfaces displayed a 36-54% increase on average in initial cell attachment, which corresponded to a more than 2-fold increase in FA localization when compared to other Silk Film surfaces and controls. In addition, distinct localization of FA formation was observed along the edges for all patterned Silk Film topographies. In conclusion, Silk Film feature topography appears to help direct corneal epithelial cell response and cytoskeleton development, especially with regard to FA distribution, in vitro.
-
Silk Film culture system for in vitro analysis and biomaterial design.
Journal of visualized experiments : JoVE, 2012Co-Authors: Brian Lawrence, David L Kaplan, Zhi Pan, Michael D. Weber, Mark I. RosenblattAbstract:Silk Films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment (1,2). These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications (3-8). In addition, Silk Films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications (9-13). The culture system methodology presented here represents a scalable approach for rapid assessments of cell-Silk Film surface interactions. Of particular interest is the use of surface patterned Silk Films to study differences in cell proliferation and responses of cells for alignment (12,14). The seeded cultures were cultured on both micro-patterned and flat Silk Film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the Silk Film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.