Search

After traumatic brain injury, the brain extracellular matrix undergoes structural rearrangement due to changes in matrix composition, activation of proteases, and deposition of chondroitin sulfate proteoglycans by reactive astrocytes to produce the glial scar. These changes lead to a softening of the tissue, where the stiffness of the contusion core and peripheral pericontusional regions becomes softer than that of healthy tissue. Pioneering mechanotransduction studies have shown that soft substrates upregulate intermediate filament proteins in reactive astrocytes; however, many other aspects of astrocyte biology remain unclear. Here, we developed a platform for the culture of cortical astrocytes using polyacrylamide (PA) gels of varying stiffness (measured in Pascal; Pa) to mimic injury-related regions in order to investigate the effects of tissue stiffness on astrocyte reactivity and morphology. Our results show that substrate stiffness influences astrocyte phenotype; soft 300 Pa substrates led to increased GFAP immunoreactivity, proliferation, and complexity of processes. Intermediate 800 Pa substrates increased Aggrecan+, Brevican+, and Neurocan+ astrocytes. The stiffest 1 kPa substrates led to astrocytes with basal morphologies, similar to a physiological state. These results advance our understanding of astrocyte mechanotransduction processes and provide evidence of how substrates with engineered stiffness can mimic the injury microenvironment.

The toxic side effects of chemotherapy have long limited its efficacy, prompting expensive and long-drawn efforts to develop more targeted cancer therapeutics. An alternative approach to mitigate off-target toxicity is to develop a device that can sequester chemotherapeutic agents from the veins that drain the target organ before they enter systemic circulation. This effectively localizes the chemotherapy to the target organ, minimizing any hazardous side effects. 3D printing is ideal for fabricating these devices, as the geometric control afforded allows us to precisely dictate its hemodynamic performance in vivo. However, the existing materials compatible with 3D printing do not have drug-binding capabilities. Here, we report the stable coating of genomic DNA on a 3D-printed structure for the capture of doxorubicin. Genomic DNA is an effective chemotherapeutic-agent capture material due to the intrinsic DNA-targeting mechanism of action of these drugs. Stable DNA coatings were achieved through a combination of electrostatic interactions and ultraviolet C (UVC, 254 nm) cross-linking. These UVC cross-linked DNA coatings were extremely stable-leaching on average 100 pg of genomic DNA per mm2 of 3D-printed structure over a period of 30 min. In vitro studies of these materials in phosphate buffered saline and human serum demonstrated that they were able to capture, on average, 72 and 60 ng of doxorubicin per mm2 of structure, respectively. The stability and efficacy of these genomic DNA-coated 3D-printed materials represent a significant step forward towards the translation of these devices to clinical applications for the potential improvement of chemotherapy treatment.

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.

Direct measurement of proteins from single cells has been realized at the microscale using microfluidic channels, capillaries, and semi-enclosed microwell arrays. Although powerful, these formats are constrained, with the enclosed geometries proving cumbersome for multistage assays, including electrophoresis followed by immunoprobing. We introduce a hybrid microfluidic format that toggles between a planar microwell array and a suspension of microparticles. The planar array is stippled in a thin sheet of polyacrylamide gel, for efficient single-cell isolation and protein electrophoresis of hundreds-to-thousands of cells. Upon mechanical release, array elements become a suspension of separation-encoded microparticles for more efficient immunoprobing due to enhanced mass transfer. Dehydrating microparticles offer improved analytical sensitivity owing to in-gel concentration of fluorescence signal for high-throughput single-cell targeted proteomics.

A nanolipoprotein particle (NLP) is a lipid bilayer disc stabilized by two amphipathic "scaffold" apolipoproteins. It has been most notably utilized as a tool for solubilizing a variety of membrane proteins while preserving structural and functional properties. Transfer of functional proteins from NLPs into model membrane systems such as supported lipid bilayers (SLBs) would enable new opportunities, for example, two-dimensional protein crystallization and studies on protein-protein interactions. This work used fluorescence microscopy and atomic force microscopy to investigate the interaction between NLPs and SLBs. When incubated with SLBs, NLPs were found to spontaneously deliver lipid and protein cargo. The impact of membrane composition on lipid exchange was explored, revealing a positive correlation between the magnitude of lipid transfer and concentration of defects in the target SLB. Incorporation of lipids capable of binding specifically to polyhistidine tags encoded into the apolipoproteins also boosted transfer of NLP cargo. Optimal conditions for lipid and protein delivery from NLPs to SLBs are proposed based on interaction mechanisms.

A nanolipoprotein particle (NLP) is a lipid bilayer disc stabilized by two amphipathic "scaffold" apolipoproteins. It has been most notably utilized as a tool for solubilizing a variety of membrane proteins while preserving structural and functional properties. Transfer of functional proteins from NLPs into model membrane systems such as supported lipid bilayers (SLBs) would enable new opportunities, for example, two-dimensional protein crystallization and studies on protein-protein interactions. This work used fluorescence microscopy and atomic force microscopy to investigate the interaction between NLPs and SLBs. When incubated with SLBs, NLPs were found to spontaneously deliver lipid and protein cargo. The impact of membrane composition on lipid exchange was explored, revealing a positive correlation between the magnitude of lipid transfer and concentration of defects in the target SLB. Incorporation of lipids capable of binding specifically to polyhistidine tags encoded into the apolipoproteins also boosted transfer of NLP cargo. Optimal conditions for lipid and protein delivery from NLPs to SLBs are proposed based on interaction mechanisms.

Embryonic stem cells (ESC) are excellent cell culture systems for elucidating developmental signals that may be part of the stem cell niche. Although stem cells are traditionally induced using predominately soluble signals, the mechanical environment of the niche can also play a role in directing cells towards differential cell lineages. Interested in diverging vascular fates, we set out to examine to what extent mechanical signaling played a role in endothelial cell and/or smooth muscle fate. Using chemically-defined staged vascular differentiation methods, vascular progenitor cells (VPC) fate was examined on single stiffness polyacrylamide hydrogels of 10 kPa, 40 kPa and >0.1 GPa. Emergence of vascular cell populations aligned with corresponding hydrogel stiffness: EC-lineages favoring the softer material and SMC lineages favoring the stiffest material. Statistical significance was observed on both cell lines on almost all days. Transcriptome analysis indicated that the populations on the varying stiffness emerge in distinct categories. Lastly, blocking studies show that αvβ1, and not αvβ6, activation mediates stiffness-directed vascular differentiation. Overall, these studies indicate that softer materials direct VPCs into a more EC-like fate compared to stiffer materials. STATEMENT OF SIGNIFICANCE: Although stem cells are traditionally induced using predominately soluble signals, the mechanical environment of the niche also plays a role in directing cell fate. Several studies have examined the stiffness-induced cell fate from mesenchymal stem cells (MSCs) and undifferentiated embryonic stem cells (ESCs). This is the first study that rigorously examines the role of matrix stiffness in diverging vascular fates from a purified population of vascular progenitor cells (VPCs). We show that the emergence of endothelial cell (EC) versus smooth muscle cell (SMC) populations corresponds with hydrogel stiffness: EC-lineages favoring the softness material and SMC lineages favoring the stiffest material, and that αvβ1 activation mediates this stiffness-directed vascular differentiation.

Embryonic stem cells (ESC) are excellent cell culture systems for elucidating developmental signals that may be part of the stem cell niche. Although stem cells are traditionally induced using predominately soluble signals, the mechanical environment of the niche can also play a role in directing cells towards differential cell lineages. Interested in diverging vascular fates, we set out to examine to what extent mechanical signaling played a role in endothelial cell and/or smooth muscle fate. Using chemically-defined staged vascular differentiation methods, vascular progenitor cells (VPC) fate was examined on single stiffness polyacrylamide hydrogels of 10 kPa, 40 kPa and >0.1 GPa. Emergence of vascular cell populations aligned with corresponding hydrogel stiffness: EC-lineages favoring the softer material and SMC lineages favoring the stiffest material. Statistical significance was observed on both cell lines on almost all days. Transcriptome analysis indicated that the populations on the varying stiffness emerge in distinct categories. Lastly, blocking studies show that αvβ1, and not αvβ6, activation mediates stiffness-directed vascular differentiation. Overall, these studies indicate that softer materials direct VPCs into a more EC-like fate compared to stiffer materials. STATEMENT OF SIGNIFICANCE: Although stem cells are traditionally induced using predominately soluble signals, the mechanical environment of the niche also plays a role in directing cell fate. Several studies have examined the stiffness-induced cell fate from mesenchymal stem cells (MSCs) and undifferentiated embryonic stem cells (ESCs). This is the first study that rigorously examines the role of matrix stiffness in diverging vascular fates from a purified population of vascular progenitor cells (VPCs). We show that the emergence of endothelial cell (EC) versus smooth muscle cell (SMC) populations corresponds with hydrogel stiffness: EC-lineages favoring the softness material and SMC lineages favoring the stiffest material, and that αvβ1 activation mediates this stiffness-directed vascular differentiation.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Five potentially environmentally friendly and light-stable hemicyanine dyes were designed based on integrated consideration of photo, environmental, and computational chemistry as well as textile applications. Two of them were synthesized and applied in dyeing polyacrylonitrile (PAN), cotton, and nylon fabrics, and demonstrated the desired properties speculated by the programs. The computer-assisted analytical processes includes estimation of the maximum absorption and emission wavelengths, aquatic environmental toxicity, affinity to fibers, and photo-stability. This procedure could effectively narrow down discovery of new potential dye structures, greatly reduce and prevent complex and expensive preparation processes, and significantly improve the development efficiency of novel environmentally friendly dyes.

Poly(lactic-co-glycolic acid) (PLGA) is widely used as a vehicle for delivery of pharmaceutically relevant payloads. PLGA is readily fabricated as a nano- or microparticle (MP) matrix to load both hydrophobic and hydrophilic small molecular drugs as well as biomacromolecules such as nucleic acids and proteins. However, targeting such payloads to the cell cytosol is often limited by MP entrapment and degradation within acidic endolysosomes. Poly(propylacrylic acid) (PPAA) is a polyelectrolyte polymer with the membrane disruptive capability triggered at low pH. PPAA has been previously formulated in various carrier configurations to enable cytosolic payload delivery, but requires sophisticated carrier design. Taking advantage of PPAA functionality, we have incorporated PPAA into PLGA MPs as a simple polymer mixture to enhance cytosolic delivery of PLGA-encapsulated payloads. Rhodamine loaded PLGA and PPAA/PLGA blend MPs were prepared by a modified nanoprecipitation method. Incorporation of PPAA into PLGA MPs had little to no effect on the size, shape, or loading efficiency, and evidenced no toxicity in Chinese hamster ovary epithelial cells. Notably, incorporation of PPAA into PLGA MPs enabled pH-dependent membrane disruption in a hemolysis assay, and a three-fold increased endosomal escape and cytosolic delivery in dendritic cells after 2 h of MP uptake. These results demonstrate that a simple PLGA/PPAA polymer blend is readily fabricated into composite MPs, enabling cytosolic delivery of an encapsulated payload. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1022-1033, 2018.

Hydrogels have shown great potential for cartilage tissue engineering applications due to their capability to encapsulate cells within biomimetic, 3-dimensional (3D) microenvironments. However, the multi-step fabrication process that is necessary to produce cell/scaffold constructs with defined dimensions, limits their off-the-shelf translational usage. In this study, we have developed a hybrid scaffolding system which combines a thermosensitive hydrogel, poly(ethylene glycol)-poly(N-isopropylacrylamide) (PEG-PNIPAAm), with a biodegradable polymer, poly(ε-caprolactone) (PCL), into a composite, electrospun microfibrous structure. A judicious optimization of material composition and electrospinning process produced a structurally self-supporting hybrid scaffold. The reverse thermosensitivity of PEG-PNIPAAm allowed its dissolution/hydration upon cell seeding within a network of PCL microfibers while maintaining the overall scaffold shape at room temperature. A subsequent temperature elevation to 37 °C induced the hydrogel's phase transition to a gel state, effectively encapsulating cells in a 3D hydrogel without the use of a mold. We demonstrated that the hybrid scaffold enhanced chondrogenic differentiation of human mesenchymal stem cells (hMSCs) based on chondrocytic gene and protein expression, which resulted in superior viscoelastic properties of the cell/scaffold constructs. The hybrid scaffold enables a facile, single-step cell seeding process to inoculate cells within a 3D hydrogel with the potential for cartilage tissue engineering.Statement of significanceHydrogels have demonstrated the excellent ability to enhance chondrogenesis of stem cells due to their hydrated fibrous nanostructure providing a cellular environment similar to native cartilage. However, the necessity for multi-step processes, including mixing of hydrogel precursor with cells and subsequent gelation in a mold to form a defined shape, limits their off-the-shelf usage. In this study, we developed a hybrid scaffold by combining a thermosensitive hydrogel with a mechanically stable polymer, which provides a facile means to inoculate cells in a 3D hydrogel with a mold-less, single step cell seeding process. We further showed that the hybrid scaffold enhanced chondrogenesis of mesenchymal stem cells, demonstrating its potential for cartilage tissue engineering.

Hydrogels have shown great potential for cartilage tissue engineering applications due to their capability to encapsulate cells within biomimetic, 3-dimensional (3D) microenvironments. However, the multi-step fabrication process that is necessary to produce cell/scaffold constructs with defined dimensions, limits their off-the-shelf translational usage. In this study, we have developed a hybrid scaffolding system which combines a thermosensitive hydrogel, poly(ethylene glycol)-poly(N-isopropylacrylamide) (PEG-PNIPAAm), with a biodegradable polymer, poly(ε-caprolactone) (PCL), into a composite, electrospun microfibrous structure. A judicious optimization of material composition and electrospinning process produced a structurally self-supporting hybrid scaffold. The reverse thermosensitivity of PEG-PNIPAAm allowed its dissolution/hydration upon cell seeding within a network of PCL microfibers while maintaining the overall scaffold shape at room temperature. A subsequent temperature elevation to 37 °C induced the hydrogel's phase transition to a gel state, effectively encapsulating cells in a 3D hydrogel without the use of a mold. We demonstrated that the hybrid scaffold enhanced chondrogenic differentiation of human mesenchymal stem cells (hMSCs) based on chondrocytic gene and protein expression, which resulted in superior viscoelastic properties of the cell/scaffold constructs. The hybrid scaffold enables a facile, single-step cell seeding process to inoculate cells within a 3D hydrogel with the potential for cartilage tissue engineering.Statement of significanceHydrogels have demonstrated the excellent ability to enhance chondrogenesis of stem cells due to their hydrated fibrous nanostructure providing a cellular environment similar to native cartilage. However, the necessity for multi-step processes, including mixing of hydrogel precursor with cells and subsequent gelation in a mold to form a defined shape, limits their off-the-shelf usage. In this study, we developed a hybrid scaffold by combining a thermosensitive hydrogel with a mechanically stable polymer, which provides a facile means to inoculate cells in a 3D hydrogel with a mold-less, single step cell seeding process. We further showed that the hybrid scaffold enhanced chondrogenesis of mesenchymal stem cells, demonstrating its potential for cartilage tissue engineering.

Biologists' preeminent toolbox for separating, analyzing, and visualizing proteins is SDS-PAGE, yet recovering the proteins embedded in these polyacrylamide media as intact species is a long-standing challenge for mass spectrometry. In conventional workflows, protein mixtures from crude biological samples are electrophoretically separated at high-resolution within N,N'-methylene-bis-acrylamide cross-linked polyacrylamide gels to reduce sample complexity and facilitate sensitive characterization. However, low protein recoveries, especially for high molecular weight proteins, often hinder characterization by mass spectrometry. We describe a workflow for top-down/bottom-up mass spectrometric analyses of proteins in polyacrylamide slab gels using dissolvable, bis-acryloylcystamine-cross-linked polyacrylamide, enabling high-resolution protein separations while recovering intact proteins over a broad size range efficiently. The inferior electrophoretic resolution long associated with reducible gels has been overcome, as demonstrated by SDS-PAGE of crude tissue extracts. This workflow elutes intact proteins efficiently, supporting MS and MS/MS from proteins resolved on biologists' preferred separation platform.

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell-cell adhesion.

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell-cell adhesion.

Targeted delivery of anti-inflammatory osteoarthritis treatments have the potential to significantly decrease undesirable systemic side effects and reduce required therapeutic dosage. Here we present a targeted, non-invasive drug delivery system to decrease inflammation in an osteoarthritis model. Hollow thermoresponsive poly(N-isopropylacrylamide) (pNIPAM) nanoparticles have been synthesized via degradation of a N,N'-bis(acryloyl)cystamine (BAC) cross-linked core out of a non-degradable pNIPAM shell. Sulfated 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) was copolymerized in the shell to increase passive loading of an anti-inflammatory mitogen-activated protein kinase-activated protein kinase 2 (MK2)-inhibiting cell-penetrating peptide (KAFAK). The drug-loaded hollow nanoparticles were effective at delivering a therapeutically active dose of KAFAK to bovine cartilage explants, suppressing pro-inflammatory interleukin-6 (IL-6) expression after interleukin-1 beta (IL-1β) stimulation. This thermosensitive hollow nanoparticle system provides an excellent platform for the delivery of peptide therapeutics into highly proteolytic environments such as osteoarthritis.

The spatial presentation of mechanical information is a key parameter for cell behavior. We have developed a method of polymerization control in which the differential diffusion distance of unreacted cross-linker and monomer into a prepolymerized hydrogel sink results in a tunable stiffness gradient at the cell-matrix interface. This simple, low-cost, robust method was used to produce polyacrylamide hydrogels with stiffness gradients of 0.5, 1.7, 2.9, 4.5, 6.8, and 8.2 kPa/mm, spanning the in vivo physiological and pathological mechanical landscape. Importantly, three of these gradients were found to be nondurotactic for human adipose-derived stem cells (hASCs), allowing the presentation of a continuous range of stiffnesses in a single well without the confounding effect of differential cell migration. Using these nondurotactic gradient gels, stiffness-dependent hASC morphology, migration, and differentiation were studied. Finally, the mechanosensitive proteins YAP, Lamin A/C, Lamin B, MRTF-A, and MRTF-B were analyzed on these gradients, providing higher-resolution data on stiffness-dependent expression and localization.

The spatial presentation of mechanical information is a key parameter for cell behavior. We have developed a method of polymerization control in which the differential diffusion distance of unreacted cross-linker and monomer into a prepolymerized hydrogel sink results in a tunable stiffness gradient at the cell-matrix interface. This simple, low-cost, robust method was used to produce polyacrylamide hydrogels with stiffness gradients of 0.5, 1.7, 2.9, 4.5, 6.8, and 8.2 kPa/mm, spanning the in vivo physiological and pathological mechanical landscape. Importantly, three of these gradients were found to be nondurotactic for human adipose-derived stem cells (hASCs), allowing the presentation of a continuous range of stiffnesses in a single well without the confounding effect of differential cell migration. Using these nondurotactic gradient gels, stiffness-dependent hASC morphology, migration, and differentiation were studied. Finally, the mechanosensitive proteins YAP, Lamin A/C, Lamin B, MRTF-A, and MRTF-B were analyzed on these gradients, providing higher-resolution data on stiffness-dependent expression and localization.

Nasal tip mechanical stability is important for functional and cosmetic nasal airway surgery. Palpation of the nasal tip provides information on tip strength to the surgeon, though it is a purely subjective assessment. Providing a means to simulate nasal tip deformation with a validated model can offer a more objective approach in understanding the mechanics and nuances of the nasal tip support and eventual nasal mechanics as a whole. Herein we present validation of a finite element (FE) model of the nose using physical measurements recorded using an ABS plastic-silicone nasal phantom. Three-dimensional photogrammetry was used to capture the geometry of the phantom at rest and while under steady state load. The silicone used to make the phantom was mechanically tested and characterized using a linear elastic constitutive model. Surface point clouds of the silicone and FE model were compared for both the loaded and unloaded state. The average Hausdorff distance between actual measurements and FE simulations across the nose were 0.39 ± 1.04 mm and deviated up to 2 mm at the outermost boundaries of the model. FE simulation and measurements were in near complete agreement in the immediate vicinity of the nasal tip with millimeter accuracy. We have demonstrated validation of a two-component nasal FE model, which could be used to model more complex modes of deformation where direct measurement may be challenging. This is the first step in developing a nasal model to simulate nasal mechanics and ultimately the interaction between geometry and airflow.

Nasal tip mechanical stability is important for functional and cosmetic nasal airway surgery. Palpation of the nasal tip provides information on tip strength to the surgeon, though it is a purely subjective assessment. Providing a means to simulate nasal tip deformation with a validated model can offer a more objective approach in understanding the mechanics and nuances of the nasal tip support and eventual nasal mechanics as a whole. Herein we present validation of a finite element (FE) model of the nose using physical measurements recorded using an ABS plastic-silicone nasal phantom. Three-dimensional photogrammetry was used to capture the geometry of the phantom at rest and while under steady state load. The silicone used to make the phantom was mechanically tested and characterized using a linear elastic constitutive model. Surface point clouds of the silicone and FE model were compared for both the loaded and unloaded state. The average Hausdorff distance between actual measurements and FE simulations across the nose were 0.39 ± 1.04 mm and deviated up to 2 mm at the outermost boundaries of the model. FE simulation and measurements were in near complete agreement in the immediate vicinity of the nasal tip with millimeter accuracy. We have demonstrated validation of a two-component nasal FE model, which could be used to model more complex modes of deformation where direct measurement may be challenging. This is the first step in developing a nasal model to simulate nasal mechanics and ultimately the interaction between geometry and airflow.

Novel pore-expanded mesoporous silica nanoparticles (MSNs) with pore sizes of approximately 11nm were synthesized and modified with thermoresponsive, poly(n-isopropylacrylamide) (PNIPAM) gating groups on the nanoparticle exterior surface and in addition with poly(ethylene-glycol) (PEG) within the porous interior to minimize protein adsorption. PEG traditionally has been grafted to the nanoparticle exterior to minimize non-specific binding and interactions with the biological environment, but due to the templating mechanism of MSN synthesis, both the pore interior and nanoparticle surface can be separately modified. Here, an improved control release behavior of bovine hemoglobin (BHb) was observed after PEGylating the interior porous framework, compared to the release BHb from unmodified MSNs. This can be attributed to the reduced protein denaturation on PEGylated silica that was observed using circular dichroism spectroscopy.

Mimicking the zonal organization of native articular cartilage, which is essential for proper tissue functions, has remained a challenge. In this study, a thermoresponsive copolymer of chitosan-g-poly(N-isopropylacrylamide) (CS-g-PNIPAAm) was synthesized as a carrier of mesenchymal stem cells (MSCs) to provide a support for their proliferation and differentiation. Microengineered three-dimensional (3D) cell-laden CS-g-PNIPAAm hydrogels with different microstripe widths were fabricated to control cellular alignment and elongation in order to mimic the superficial zone of natural cartilage. Biochemical assays showed six- and sevenfold increment in secretion of glycosaminoglycans (GAGs) and total collagen from MSCs encapsulated within the synthesized hydrogel after 28 days incubation in chondrogenic medium. Chondrogenic differentiation was also verified qualitatively by histological and immunohistochemical assessments. It was found that 75 ± 6% of cells encapsulated within 50 μm wide microstripes were aligned with an aspect ratio of 2.07 ± 0.16 at day 5, which was more organized than those observed in unpatterned constructs (12 ± 7% alignment and a shape index of 1.20 ± 0.07). The microengineered constructs mimicked the cell shape and organization in the superficial zone of cartilage whiles the unpatterned one resembled the middle zone. Our results suggest that microfabrication of 3D cell-laden thermosensitive hydrogels is a promising platform for creating biomimetic structures leading to more successful multi-zonal cartilage tissue engineering. Biotechnol. Bioeng. 2017;114: 217-231. © 2016 Wiley Periodicals, Inc.

Mimicking the zonal organization of native articular cartilage, which is essential for proper tissue functions, has remained a challenge. In this study, a thermoresponsive copolymer of chitosan-g-poly(N-isopropylacrylamide) (CS-g-PNIPAAm) was synthesized as a carrier of mesenchymal stem cells (MSCs) to provide a support for their proliferation and differentiation. Microengineered three-dimensional (3D) cell-laden CS-g-PNIPAAm hydrogels with different microstripe widths were fabricated to control cellular alignment and elongation in order to mimic the superficial zone of natural cartilage. Biochemical assays showed six- and sevenfold increment in secretion of glycosaminoglycans (GAGs) and total collagen from MSCs encapsulated within the synthesized hydrogel after 28 days incubation in chondrogenic medium. Chondrogenic differentiation was also verified qualitatively by histological and immunohistochemical assessments. It was found that 75 ± 6% of cells encapsulated within 50 μm wide microstripes were aligned with an aspect ratio of 2.07 ± 0.16 at day 5, which was more organized than those observed in unpatterned constructs (12 ± 7% alignment and a shape index of 1.20 ± 0.07). The microengineered constructs mimicked the cell shape and organization in the superficial zone of cartilage whiles the unpatterned one resembled the middle zone. Our results suggest that microfabrication of 3D cell-laden thermosensitive hydrogels is a promising platform for creating biomimetic structures leading to more successful multi-zonal cartilage tissue engineering. Biotechnol. Bioeng. 2017;114: 217-231. © 2016 Wiley Periodicals, Inc.

Novel pore-expanded mesoporous silica nanoparticles (MSNs) with pore sizes of approximately 11nm were synthesized and modified with thermoresponsive, poly(n-isopropylacrylamide) (PNIPAM) gating groups on the nanoparticle exterior surface and in addition with poly(ethylene-glycol) (PEG) within the porous interior to minimize protein adsorption. PEG traditionally has been grafted to the nanoparticle exterior to minimize non-specific binding and interactions with the biological environment, but due to the templating mechanism of MSN synthesis, both the pore interior and nanoparticle surface can be separately modified. Here, an improved control release behavior of bovine hemoglobin (BHb) was observed after PEGylating the interior porous framework, compared to the release BHb from unmodified MSNs. This can be attributed to the reduced protein denaturation on PEGylated silica that was observed using circular dichroism spectroscopy.

Polyacrylamide hydrogels have been used extensively to study cell responses to the mechanical and biochemical properties of extracellular matrix substrates. A key step in fabricating these substrates is the conjugation of cell adhesion proteins to the polyacrylamide surfaces, which typically involves nonspecifically anchoring these proteins via side-chain functional groups. This can result in a loss of presentation control and altered bioactivity. Here, we describe a new functionalization strategy in which we anchor full-length extracellular matrix proteins to polyacrylamide substrates using 2-pyridinecarboxaldehyde, which can be co-polymerized into polyacrylamide gels and used to immobilize proteins by their N-termini. This one-step reaction proceeds under mild aqueous conditions and does not require additional reagents. We demonstrate that these substrates can readily conjugate to various extracellular matrix proteins, as well as promote cell adhesion and spreading. Notably, this chemistry supports the assembly and cellular remodeling of large collagen fibers, which is not observed using conventional side-chain amine-conjugation chemistry.

Purpose The purpose of this study was to (1) develop a novel tissue-mimicking thermochromic (TMTC) phantom that permanently changes colour from white to magenta upon heating above ablative temperatures, and (2) assess its utility for specific applications in evaluating thermal therapy devices. Materials and methods Polyacrylamide gel mixed with thermochromic ink was custom made to produce a TMTC phantom that changes its colour upon heating above biological ablative temperatures (> 60 °C). The thermal properties of the phantom were characterised, and compared to those of human tissue. In addition, utility of this phantom as a tool for the assessment of laser and microwave thermal ablation was examined. Results The mass density, thermal conductivity, and thermal diffusivity of the TMTC phantom were measured as 1033 ± 1.0 kg/m(3), 0.590 ± 0.015 W/m.K, and 0.145 ± 0.002 mm(2)/s, respectively, and found to be in agreement with reported values for human soft tissues. Heating the phantom with laser and microwave ablation devices produced clearly demarcated regions of permanent colour change geographically corresponding to regions with temperature elevations above 60 °C. Conclusion The TMTC phantom provides direct visualisation of ablation dynamics, including ablation volume and geometry as well as peak absolute temperatures within the treated region post-ablation. This phantom can be specifically tailored for different thermal therapy modalities, such as radiofrequency, laser, microwave, or therapeutic ultrasound ablation. Such modality-specific phantoms may enable better quality assurance, device characterisation, and ablation parameter optimisation, or optimise the study of dynamic heating parameters integral to drug device combination therapies relying upon heat.

Topical administration of drugs and growth factors in a controlled fashion can improve the healing process during skin disorders and chronic wounds. To achieve this goal, a dermal patch is engineered that utilizes thermoresponsive drug microcarriers encapsulated within a hydrogel layer attached to a flexible heater with integrated electronic heater control circuitry. The engineered patch conformally covers the wound area and enables controlled drug delivery by electronically adjusting the temperature of the hydrogel layer. The drugs are encapsulated inside microparticles in order to control their release rates. These monodisperse thermoresponsive microparticles containing active molecules are fabricated using a microfluidic device. The system is used to release two different active molecules with molecular weights similar to drugs and growth factors and their release profiles are characterized. This platform is a key step towards engineering smart and closed loop systems for topical applications.

Topical administration of drugs and growth factors in a controlled fashion can improve the healing process during skin disorders and chronic wounds. To achieve this goal, a dermal patch is engineered that utilizes thermoresponsive drug microcarriers encapsulated within a hydrogel layer attached to a flexible heater with integrated electronic heater control circuitry. The engineered patch conformally covers the wound area and enables controlled drug delivery by electronically adjusting the temperature of the hydrogel layer. The drugs are encapsulated inside microparticles in order to control their release rates. These monodisperse thermoresponsive microparticles containing active molecules are fabricated using a microfluidic device. The system is used to release two different active molecules with molecular weights similar to drugs and growth factors and their release profiles are characterized. This platform is a key step towards engineering smart and closed loop systems for topical applications.

Core-shell nanoparticles comprised of Fe3O4 cores and a mesoporous silica shell with an average expanded pore size of 6.07 nm and coated with a poly(N-isopropylacrylamide) (PNIPAM) layer (CS-MSNs-EP-PNIPAM) were prepared and characterized. The nanoparticles was loaded with (Ru(bipy)3(2+)) dye or an antibacterial enzyme, lysozyme, to obtain CS-MSNs-EP-PNIPAM-Ru(bipy)3(2+) and CS-MSNs-EP-PNIPAM-Lys, respectively. The lysozyme loading was determined to be 160 mg/g of nanoparticle. It was seen that Ru(bipy)3(2+) and lysozyme release was minimal at a room temperature of 25 °C while at physiological temperature (37 °C), abrupt release was observed. The applicability of the CS-MSNs-EP-PNIPAM-Lys was further tested with two Gram-positive bacteria samples, Bacillus cereus and Micrococcus luteus. At physiological temperature, the nanoparticles were shown to reduce bacterial growth, indicating a successful release of lysozyme from the nanoparticles. This nanoparticle system shows potential as a nanocarrier for the loading of similarly sized proteins or other species as a drug delivery platform.

Core-shell nanoparticles comprised of Fe3O4 cores and a mesoporous silica shell with an average expanded pore size of 6.07 nm and coated with a poly(N-isopropylacrylamide) (PNIPAM) layer (CS-MSNs-EP-PNIPAM) were prepared and characterized. The nanoparticles was loaded with (Ru(bipy)3(2+)) dye or an antibacterial enzyme, lysozyme, to obtain CS-MSNs-EP-PNIPAM-Ru(bipy)3(2+) and CS-MSNs-EP-PNIPAM-Lys, respectively. The lysozyme loading was determined to be 160 mg/g of nanoparticle. It was seen that Ru(bipy)3(2+) and lysozyme release was minimal at a room temperature of 25 °C while at physiological temperature (37 °C), abrupt release was observed. The applicability of the CS-MSNs-EP-PNIPAM-Lys was further tested with two Gram-positive bacteria samples, Bacillus cereus and Micrococcus luteus. At physiological temperature, the nanoparticles were shown to reduce bacterial growth, indicating a successful release of lysozyme from the nanoparticles. This nanoparticle system shows potential as a nanocarrier for the loading of similarly sized proteins or other species as a drug delivery platform.

Antimicrobial peptides (AMPs) are good candidates to treat burn wounds, a major cause of morbidity, impaired life quality and resources consumption in developed countries. We took advantage of a commercially available hydrogel, Carbopol®, a vehicle for topical administration that maintains a moist environment within the wound site. We hypothesized that the incorporation of LLKKK18 conjugated to dextrin would improve the healing process in rat burns. Whereas the hydrogel improves healing, LLKKK18 released from the dextrin conjugates further accelerated wound closure, and simultaneously improving the quality of healing. Indeed, the release of LLKKK18 reduced oxidative stress and inflammation (low neutrophil and macrophage infiltration and pro-inflammatory cytokines levels). Importantly, it induced a faster resolution of the inflammatory stage through early M2 macrophage recruitment. In addition, LLKKK18 stimulated angiogenesis (increased VEGF and microvessel development in vivo). Moreover, collagen staining evaluated by Masson's Trichrome was visually much more intense after treatment with LLKKK18, suggesting higher collagen deposition. Overall, we generated an effective, safe and inexpensive formulation that maintains a moist environment in the wound, easy to apply and remove, and with potential to prevent infection due to the presence of an antimicrobial peptide. These findings propel us to further study this LLKKK18-containing formulation, setting the foundations towards a potential therapeutic approach for burn wound treatment.Statement of significanceThis work presents a newly developed formulation that holds great potential as a therapeutic approach for burn treatment. It is based on the sustained delivery of an antimicrobial peptide - LLKKK18 - from conjugates with dextrin, after degradation of dextrin backbone upon exposure to wound α-amylases. Conjugates were further embedded in Carbopol®, a commercially available hydrogel, suitable for topical administration and that provides a moist environment to the wound. Overall, we obtained an efficient, safe and non-expensive formulation that improves burn wound healing, maintains a moist environment within the wound, is easy to apply-and-remove, and has potential to prevent infection due to the presence of an antimicrobial peptide. Importantly, this is the first time the wound healing ability of LLKKK18 is demonstrated and that its main mechanisms of action are identified.

UnlabelledAntimicrobial peptides (AMPs) are good candidates to treat burn wounds, a major cause of morbidity, impaired life quality and resources consumption in developed countries. We took advantage of a commercially available hydrogel, Carbopol®, a vehicle for topical administration that maintains a moist environment within the wound site. We hypothesized that the incorporation of LLKKK18 conjugated to dextrin would improve the healing process in rat burns. Whereas the hydrogel improves healing, LLKKK18 released from the dextrin conjugates further accelerated wound closure, and simultaneously improving the quality of healing. Indeed, the release of LLKKK18 reduced oxidative stress and inflammation (low neutrophil and macrophage infiltration and pro-inflammatory cytokines levels). Importantly, it induced a faster resolution of the inflammatory stage through early M2 macrophage recruitment. In addition, LLKKK18 stimulated angiogenesis (increased VEGF and microvessel development in vivo). Moreover, collagen staining evaluated by Masson's Trichrome was visually much more intense after treatment with LLKKK18, suggesting higher collagen deposition. Overall, we generated an effective, safe and inexpensive formulation that maintains a moist environment in the wound, easy to apply and remove, and with potential to prevent infection due to the presence of an antimicrobial peptide. These findings propel us to further study this LLKKK18-containing formulation, setting the foundations towards a potential therapeutic approach for burn wound treatment.Statement of significanceThis work presents a newly developed formulation that holds great potential as a therapeutic approach for burn treatment. It is based on the sustained delivery of an antimicrobial peptide - LLKKK18 - from conjugates with dextrin, after degradation of dextrin backbone upon exposure to wound α-amylases. Conjugates were further embedded in Carbopol®, a commercially available hydrogel, suitable for topical administration and that provides a moist environment to the wound. Overall, we obtained an efficient, safe and non-expensive formulation that improves burn wound healing, maintains a moist environment within the wound, is easy to apply-and-remove, and has potential to prevent infection due to the presence of an antimicrobial peptide. Importantly, this is the first time the wound healing ability of LLKKK18 is demonstrated and that its main mechanisms of action are identified.

Peripheral artery disease is an atherosclerotic occlusion in the peripheral vasculature that is typically treated via percutaneous transluminal angioplasty. Unfortunately, deployment of the angioplasty balloon damages the endothelial layer, exposing the underlying collagen and allowing for the binding and activation of circulating platelets, which initiate an inflammatory cascade leading to eventual restenosis. Here, we report on the development of poly(NIPAm-MBA-AMPS-AAc) nanoparticles that have a collagen I-binding peptide crosslinked to their surface allowing them to bind to exposed collagen. Once bound, these particles mask the exposed collagen from circulating platelets, effectively reducing collagen-mediated platelet activation. Using collagen I-coated plates, we demonstrate that these particles are able to bind to collagen at concentrations above 0.5 mg/mL. Once bound, these particles inhibit collagen-mediated platelet activation by over 60%. Using light scattering and zeta potential measurements, we investigated the potential of the nanoparticles as a drug delivery platform. We have verified that the collagen-binding nanoparticles retain the temperature sensitivity common to poly(NIPAm)-based nanoparticles while remaining colloidally stable in aqueous environments. We also demonstrate that they are able to passively load and release anti-inflammatory cell penetrating peptides. Combined, we have developed a collagen-binding nanoparticle that has dual therapy potential, preventing collagen-mediated platelet activation while delivering water-soluble therapeutics directly to the damaged area.

The semen-derived enhancer of virus infection (SEVI) is a natural amyloid material that has been shown to substantially increase viral attachment and infectivity of HIV in cells. We previously reported that synthetic monomeric and oligomeric amyloid-targeting molecules could form protein-resistive coatings on SEVI and inhibit SEVI- and semen-mediated enhancement of HIV infectivity. While oligomeric amyloid-binding compounds showed substantial improvement in apparent binding to SEVI compared to monomeric compounds, we observed only a modest correlation between apparent binding to SEVI and activity for reducing SEVI-mediated HIV infection. Here, we synthesized amyloid-binding polyacrylate-based polymers and polymeric nanoparticles of comparable size to HIV virus particles (∼150 nm) to assess the effect of sterics on the inhibition of SEVI-mediated enhancement of HIV infectivity. We show that these polymeric materials exhibit excellent capability to reduce SEVI-mediated enhancement of HIV infection, with the nanoparticles exhibiting the greatest activity (IC50 value of ∼4 μg/mL, or 59 nM based on polymer) of any SEVI-neutralizing agent reported to date. The results support that the improved activity of these nanomaterials is likely due to their increased size (diameters = 80-200 nm) compared to amyloid-targeting small molecules and that steric interactions may play as important a role as binding affinity in inhibiting viral infection mediated by SEVI amyloids. In contrast to the previously reported SEVI-neutralizing, amyloid-targeting molecules (which required concentrations at least 100-fold above the Kd to observe activity), the approximate 1:1 ratio of apparent Kd to IC50 for activity of these polymeric materials suggests the majority of polymer molecules that are bound to SEVI contribute to the inhibition of HIV infectivity enhanced by SEVI. Such size-related effects on physical inhibition of protein-protein interactions may open further opportunities for the use of targeted nanomaterials in disease intervention.

The semen-derived enhancer of virus infection (SEVI) is a natural amyloid material that has been shown to substantially increase viral attachment and infectivity of HIV in cells. We previously reported that synthetic monomeric and oligomeric amyloid-targeting molecules could form protein-resistive coatings on SEVI and inhibit SEVI- and semen-mediated enhancement of HIV infectivity. While oligomeric amyloid-binding compounds showed substantial improvement in apparent binding to SEVI compared to monomeric compounds, we observed only a modest correlation between apparent binding to SEVI and activity for reducing SEVI-mediated HIV infection. Here, we synthesized amyloid-binding polyacrylate-based polymers and polymeric nanoparticles of comparable size to HIV virus particles (∼150 nm) to assess the effect of sterics on the inhibition of SEVI-mediated enhancement of HIV infectivity. We show that these polymeric materials exhibit excellent capability to reduce SEVI-mediated enhancement of HIV infection, with the nanoparticles exhibiting the greatest activity (IC50 value of ∼4 μg/mL, or 59 nM based on polymer) of any SEVI-neutralizing agent reported to date. The results support that the improved activity of these nanomaterials is likely due to their increased size (diameters = 80-200 nm) compared to amyloid-targeting small molecules and that steric interactions may play as important a role as binding affinity in inhibiting viral infection mediated by SEVI amyloids. In contrast to the previously reported SEVI-neutralizing, amyloid-targeting molecules (which required concentrations at least 100-fold above the Kd to observe activity), the approximate 1:1 ratio of apparent Kd to IC50 for activity of these polymeric materials suggests the majority of polymer molecules that are bound to SEVI contribute to the inhibition of HIV infectivity enhanced by SEVI. Such size-related effects on physical inhibition of protein-protein interactions may open further opportunities for the use of targeted nanomaterials in disease intervention.

As platelets aggregate and activate at the site of vascular injury to stem bleeding, they are subjected to a myriad of biochemical and biophysical signals and cues. As clot formation ensues, platelets interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Here, we show for the first time (to our knowledge) that platelets, which are anucleate cellular fragments, sense microenvironmental mechanical properties, such as substrate stiffness, and transduce those cues into differential biological signals. Specifically, as platelets mechanosense the stiffness of the underlying fibrin/fibrinogen substrate, increasing substrate stiffness leads to increased platelet adhesion and spreading. Importantly, adhesion on stiffer substrates also leads to higher levels of platelet activation, as measured by integrin αIIbβ3 activation, α-granule secretion, and procoagulant activity. Mechanistically, we determined that Rac1 and actomyosin activity mediate substrate stiffness-dependent platelet adhesion, spreading, and activation to different degrees. This capability of platelets to mechanosense microenvironmental cues in a growing thrombus or hemostatic plug and then mechanotransduce those cues into differential levels of platelet adhesion, spreading, and activation provides biophysical insight into the underlying mechanisms of platelet aggregation and platelet activation heterogeneity during thrombus formation.

Glioblastoma multiforme (GBM), the most prevalent primary brain cancer, is characterized by diffuse infiltration of tumor cells into brain tissue, which severely complicates surgical resection and contributes to tumor recurrence. The most rapid mode of tissue infiltration occurs along blood vessels or white matter tracts, which represent topological interfaces thought to serve as "tracks" that speed cell migration. Despite this observation, the field lacks experimental paradigms that capture key features of these tissue interfaces and allow reductionist dissection of mechanisms of this interfacial motility. To address this need, we developed a culture system in which tumor cells are sandwiched between a fibronectin-coated ventral surface representing vascular basement membrane and a dorsal hyaluronic acid (HA) surface representing brain parenchyma. We find that inclusion of the dorsal HA surface induces formation of adhesive complexes and significantly slows cell migration relative to a free fibronectin-coated surface. This retardation is amplified by inclusion of integrin binding peptides in the dorsal layer and expression of CD44, suggesting that the dorsal surface slows migration through biochemically specific mechanisms rather than simple steric hindrance. Moreover, both the reduction in migration speed and assembly of dorsal adhesions depend on myosin activation and the stiffness of the ventral layer, implying that mechanochemical feedback directed by the ventral layer can influence adhesive signaling at the dorsal surface.

Glioblastoma multiforme (GBM), the most prevalent primary brain cancer, is characterized by diffuse infiltration of tumor cells into brain tissue, which severely complicates surgical resection and contributes to tumor recurrence. The most rapid mode of tissue infiltration occurs along blood vessels or white matter tracts, which represent topological interfaces thought to serve as "tracks" that speed cell migration. Despite this observation, the field lacks experimental paradigms that capture key features of these tissue interfaces and allow reductionist dissection of mechanisms of this interfacial motility. To address this need, we developed a culture system in which tumor cells are sandwiched between a fibronectin-coated ventral surface representing vascular basement membrane and a dorsal hyaluronic acid (HA) surface representing brain parenchyma. We find that inclusion of the dorsal HA surface induces formation of adhesive complexes and significantly slows cell migration relative to a free fibronectin-coated surface. This retardation is amplified by inclusion of integrin binding peptides in the dorsal layer and expression of CD44, suggesting that the dorsal surface slows migration through biochemically specific mechanisms rather than simple steric hindrance. Moreover, both the reduction in migration speed and assembly of dorsal adhesions depend on myosin activation and the stiffness of the ventral layer, implying that mechanochemical feedback directed by the ventral layer can influence adhesive signaling at the dorsal surface.

As platelets aggregate and activate at the site of vascular injury to stem bleeding, they are subjected to a myriad of biochemical and biophysical signals and cues. As clot formation ensues, platelets interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Here, we show for the first time (to our knowledge) that platelets, which are anucleate cellular fragments, sense microenvironmental mechanical properties, such as substrate stiffness, and transduce those cues into differential biological signals. Specifically, as platelets mechanosense the stiffness of the underlying fibrin/fibrinogen substrate, increasing substrate stiffness leads to increased platelet adhesion and spreading. Importantly, adhesion on stiffer substrates also leads to higher levels of platelet activation, as measured by integrin αIIbβ3 activation, α-granule secretion, and procoagulant activity. Mechanistically, we determined that Rac1 and actomyosin activity mediate substrate stiffness-dependent platelet adhesion, spreading, and activation to different degrees. This capability of platelets to mechanosense microenvironmental cues in a growing thrombus or hemostatic plug and then mechanotransduce those cues into differential levels of platelet adhesion, spreading, and activation provides biophysical insight into the underlying mechanisms of platelet aggregation and platelet activation heterogeneity during thrombus formation.

The analysis of atomic force microscopy (AFM) force data requires the selection of a contact point (CP) and is often time consuming and subjective due to influence from intermolecular forces and low signal-to-noise ratios (SNR). In this report, we present an automated algorithm for the selection of CPs in AFM force data and the evaluation of elastic moduli. We propose that CP may be algorithmically easier to detect by identifying a linear elastic indentation region of data (high SNR) rather than the contact point itself (low SNR). Utilizing Hertzian mechanics, the data are fitted for the CP. We first detail the algorithm and then evaluate it on sample polymeric and biological materials. As a demonstration of automation, 64 × 64 force maps were analyzed to yield spatially varying topographical and mechanical information of cells. Finally, we compared manually selected CPs to automatically identified CPs and demonstrated that our automated approach is both accurate (< 10nm difference between manual and automatic) and precise for non-interacting polymeric materials. Our data show that the algorithm is useful for analysis of both biomaterials and biological samples.

Polymeric materials that intrinsically heal at damage sites under wet or moist conditions are urgently needed for biomedical and environmental applications. Although hydrogels with self-mending properties have been engineered by means of mussel-inspired metal-chelating catechol-functionalized polymer networks, biological self-healing in wet conditions, as occurs in self-assembled holdfast proteins in mussels and other marine organisms, is generally thought to involve more than reversible metal chelates. Here we demonstrate self-mending in metal-free water of synthetic polyacrylate and polymethacrylate materials that are surface-functionalized with mussel-inspired catechols. Wet self-mending of scission in these polymers is initiated and accelerated by hydrogen bonding between interfacial catechol moieties, and consolidated by the recruitment of other non-covalent interactions contributed by subsurface moieties. The repaired and pristine samples show similar mechanical properties, suggesting that the triggering of complete self-healing is enabled underwater by the formation of extensive catechol-mediated interfacial hydrogen bonds.

Polymeric materials that intrinsically heal at damage sites under wet or moist conditions are urgently needed for biomedical and environmental applications. Although hydrogels with self-mending properties have been engineered by means of mussel-inspired metal-chelating catechol-functionalized polymer networks, biological self-healing in wet conditions, as occurs in self-assembled holdfast proteins in mussels and other marine organisms, is generally thought to involve more than reversible metal chelates. Here we demonstrate self-mending in metal-free water of synthetic polyacrylate and polymethacrylate materials that are surface-functionalized with mussel-inspired catechols. Wet self-mending of scission in these polymers is initiated and accelerated by hydrogen bonding between interfacial catechol moieties, and consolidated by the recruitment of other non-covalent interactions contributed by subsurface moieties. The repaired and pristine samples show similar mechanical properties, suggesting that the triggering of complete self-healing is enabled underwater by the formation of extensive catechol-mediated interfacial hydrogen bonds.

ObjectivesThe aim of this study was to test the hypothesis that cross-polarization optical coherence tomography (CP-OCT) can be used to longitudinally monitor demineralization peripheral to orthodontic brackets in an extended clinical study.MethodsA high-speed CP-OCT system was used to acquire 3D volumetric images of the area at the base of orthodontic brackets over a period of 12 months after placement. The reflectivity was measured at 3-month intervals for 12 months to determine if there was increased demineralization. Two teeth were monitored on 20 test subjects and the brackets were bonded using two types of adhesives. This was a randomized controlled clinical study with a split mouth design such that each subject served as his or her own control. On one side, the control premolar was bonded with a bonding agent (Adper Scotchbond from 3M ESPE, St. Paul, MN) and composite (Transbond XT from 3M Unitek, Monrovia, CA) that lacked fluoride. On the other side, the experimental premolar was bonded with a fluoride releasing glass ionomer cement (GC Fuji Ortho LC from GC America, Alsip, IL).ResultsThere was a small but significant increase in the calculated lesion depth and integrated reflectivity over that depth (ΔR) for both adhesive types (p

Protein adsorption on nanoparticles is closely associated with the physicochemical properties of particles, in particular, their surface properties. We synthesized two batches of polyacrylic acid-coated nanoparticles under almost identical conditions except for the heating duration and found differences in the head-group structure of the polyacrylic acid. The structure change was confirmed by NMR and MS. The two batches of particles had varied binding affinities to a selected group of proteins. Computational work confirmed that the head group of the polymer on the surface of a nanoparticle could directly interact with a protein, and small structural changes in the head group were sufficient to result in a significant difference in the free energy of binding. Our results demonstrate that protein adsorption is so sensitive to the surface properties of particles that it can reveal even small variations in the structure of a nanoparticle surface ligand, and should be useful for quick assessment of nanoparticle properties.

Protein adsorption on nanoparticles is closely associated with the physicochemical properties of particles, in particular, their surface properties. We synthesized two batches of polyacrylic acid-coated nanoparticles under almost identical conditions except for the heating duration and found differences in the head-group structure of the polyacrylic acid. The structure change was confirmed by NMR and MS. The two batches of particles had varied binding affinities to a selected group of proteins. Computational work confirmed that the head group of the polymer on the surface of a nanoparticle could directly interact with a protein, and small structural changes in the head group were sufficient to result in a significant difference in the free energy of binding. Our results demonstrate that protein adsorption is so sensitive to the surface properties of particles that it can reveal even small variations in the structure of a nanoparticle surface ligand, and should be useful for quick assessment of nanoparticle properties.

Infectious bovine keratoconjunctivitis (IBK) caused by Moraxella bovis is the most common eye disease of cattle. The pathogenesis of M. bovis requires the expression of pili that enable the organism to attach to the ocular surface and an RTX (repeats in the structural toxin) toxin (cytotoxin or hemolysin), which is cytotoxic to corneal epithelial cells. In this pilot study, ocular mucosal immune responses of steers were measured following intranasal (i.n.) vaccination with a recombinant M. bovis cytotoxin adjuvanted with polyacrylic acid. Beef steers were vaccinated with either 500 μg (n = 3) or 200 μg (n = 3) of recombinant M. bovis cytotoxin plus adjuvant. Control group steers (n = 2) were vaccinated with adjuvant alone, and all steers were given a booster on day 21. Antigen-specific tear IgA and tear IgG, tear cytotoxin-neutralizing antibody responses, and serum cytotoxin-neutralizing antibody responses were determined in samples collected prevaccination and on days 14, 28, 42, and 55. Changes in tear antigen-specific IgA levels from day 0 to days 28, 42, and 55 were significantly different between groups; however, in post hoc comparisons between individual group pairs at the tested time points, the differences were not significant. Our results suggest that i.n. vaccination of cattle with recombinant M. bovis cytotoxin adjuvanted with polyacrylic acid effects changes in ocular antigen-specific IgA concentrations. The use of intranasally administered recombinant M. bovis cytotoxin adjuvanted with polyacrylic acid could provide an alternative to parenteral vaccination of cattle for immunoprophylaxis against IBK.

Infectious bovine keratoconjunctivitis (IBK) caused by Moraxella bovis is the most common eye disease of cattle. The pathogenesis of M. bovis requires the expression of pili that enable the organism to attach to the ocular surface and an RTX (repeats in the structural toxin) toxin (cytotoxin or hemolysin), which is cytotoxic to corneal epithelial cells. In this pilot study, ocular mucosal immune responses of steers were measured following intranasal (i.n.) vaccination with a recombinant M. bovis cytotoxin adjuvanted with polyacrylic acid. Beef steers were vaccinated with either 500 μg (n = 3) or 200 μg (n = 3) of recombinant M. bovis cytotoxin plus adjuvant. Control group steers (n = 2) were vaccinated with adjuvant alone, and all steers were given a booster on day 21. Antigen-specific tear IgA and tear IgG, tear cytotoxin-neutralizing antibody responses, and serum cytotoxin-neutralizing antibody responses were determined in samples collected prevaccination and on days 14, 28, 42, and 55. Changes in tear antigen-specific IgA levels from day 0 to days 28, 42, and 55 were significantly different between groups; however, in post hoc comparisons between individual group pairs at the tested time points, the differences were not significant. Our results suggest that i.n. vaccination of cattle with recombinant M. bovis cytotoxin adjuvanted with polyacrylic acid effects changes in ocular antigen-specific IgA concentrations. The use of intranasally administered recombinant M. bovis cytotoxin adjuvanted with polyacrylic acid could provide an alternative to parenteral vaccination of cattle for immunoprophylaxis against IBK.

PurposeThe transformation of fibroblasts to myofibroblasts is critical to corneal wound healing, stromal haze formation, and scarring. It has recently been demonstrated that the provision of biomimetic substratum topographic cues inhibits the progression toward the myofibroblast phenotype under the influence of transforming growth factor β1 (TGF-β1). The objective of this study was to determine the effect of another fundamental biophysical cue, substrate compliance, on TGF-β1-induced myofibroblast transformation of primary corneal cells isolated from human and rabbit corneas.MethodsHuman and rabbit corneal fibroblasts were cultured on surfaces of varying substrate compliance (4-71 kPa) and tissue culture plastic (TCP) (> 1 gigapascal [GPa]). Cells were cultured in media containing TGF-β1 at concentrations of 0, 1, or 10 ng/mL for 72 hours. RNA and protein were collected from cells cultured on polyacrylamide gels and TCP and were analyzed for the expression of α-smooth muscle actin (α-SMA), a key marker of myofibroblast transformation, using quantitative PCR, immunocytochemistry, and Western blot.ResultsCells grown on more compliant substrates demonstrated significantly reduced amounts of α-SMA mRNA compared with TCP. Immunocytochemistry and Western blot analysis determining the presence of α-SMA corroborated this finding, thus confirming a reduced transformation to the myofibroblast phenotype on more compliant substrates compared with cells on TCP in the presence of TGF-β1.ConclusionsThese data indicate that substrate compliance modulates TGF-β1-induced expression of α-SMA and thus influences myofibroblast transformation in the corneal stroma. This provides further evidence that biomimetic biophysical cues inhibit myofibroblast transformation and participate in stabilizing the native cellular phenotype.

A protein corona will be formed on nanoparticles (NPs) entering a biological matrix, which can influence particles' subsequent behaviors inside the biological systems. For proteins bound stably to the NPs, they can exhibit different association/dissociation rates. The binding kinetics could affect interaction of the NPs with cell surface receptors and possibly contribute to the outcomes of NPs uptake. In the present study, a method to differentiate the corona proteins based on their relative dissociation rates from the NPs was developed, employing flow field-flow fraction (F4) in combination with centrifugation. The proteins bound to the superparamagnetic iron oxide NPs (SPION) present in an IgG/albumin depleted serum were isolated via collection of the SPIONs by either F4 or centrifugation. They were subsequently analyzed by LC-MS/MS and identified. Because the SPION-protein complexes injected to F4 dissociated continuously under the nonequilibrium separation condition, only the proteins with slow enough dissociation rates would be collected with the NPs in the eluent of F4. However, in centrifugation, proteins with good affinity to the SPIONs were collected regardless of the dissociation rates of the complexes. In both cases, the nonbinding ones were washed off. Capillary electrophoresis and circular dichroism were employed to verify the binding situations of a few SPION-protein interactions, confirming the effectiveness of our method. Our results support that our method can screen for proteins binding to NPs with fast on-and-off rates, which should be the ones quickly exchanging with the free matrix proteins when the NPs are exposed to a new biological media. Thus, our method will be useful for investigation of the temporal profile of protein corona and its evolution in biological matrices as well as for high-throughput analysis of the dynamic feature of protein corona related to particle properties.