Topological defects produce kinks in biopolymer filament bundles.

Bundles of stiff filaments are ubiquitous in the living world, found both in the cytoskeleton and in the extracellular medium. These bundles are typically held together by smaller cross-linking molecules. We demonstrate, analytically, numerically, and experimentally, that such bundles can be kinked, that is, have localized regions of high curvature that are long-lived metastable states. We propose three possible mechanisms of kink stabilization: a difference in trapped length of the filament segments between two cross-links, a dislocation where the endpoint of a filament occurs within the bundle, and the braiding of the filaments in the bundle. At a high concentration of cross-links, the last two effects lead to the topologically protected kinked states. Finally, we explore, numerically and analytically, the transition of the metastable kinked state to the stable straight bundle.

Cell contact guidance via sensing anisotropy of network mechanical resistance.

Despite the ubiquitous importance of cell contact guidance, the signal-inducing contact guidance of mammalian cells in an aligned fibril network has defied elucidation. This is due to multiple interdependent signals that an aligned fibril network presents to cells, including, at least, anisotropy of adhesion, porosity, and mechanical resistance. By forming aligned fibrin gels with the same alignment strength, but cross-linked to different extents, the anisotropic mechanical resistance hypothesis of contact guidance was tested for human dermal fibroblasts. The cross-linking was shown to increase the mechanical resistance anisotropy, without detectable change in network microstructure and without change in cell adhesion to the cross-linked fibrin gel. This methodology thus isolated anisotropic mechanical resistance as a variable for fixed anisotropy of adhesion and porosity. The mechanical resistance anisotropy |Y*| -1 - |X*| -1 increased over fourfold in terms of the Fourier magnitudes of microbead displacement |X*| and |Y*| at the drive frequency with respect to alignment direction Y obtained by optical forces in active microrheology. Cells were found to exhibit stronger contact guidance in the cross-linked gels possessing greater mechanical resistance anisotropy: the cell anisotropy index based on the tensor of cell orientation, which has a range 0 to 1, increased by 18% with the fourfold increase in mechanical resistance anisotropy. We also show that modulation of adhesion via function-blocking antibodies can modulate the guidance response, suggesting a concomitant role of cell adhesion. These results indicate that fibroblasts can exhibit contact guidance in aligned fibril networks by sensing anisotropy of network mechanical resistance.

Clinical evaluation of a novel subcutaneous lactate monitor.

Lactate levels are commonly used as an indirect measure to assess metabolic stress in clinical conditions like sepsis. Dynamic lactate measurements are recommended to assess and guide treatment in patients with shock and other critical care conditions. A minimally invasive, continuous lactate monitor has potential to improve clinical decisions and patient care. The purpose of the study was to evaluate continuous lactate measurements of a novel enzymatic Continuous Lactate Monitor (CLM) developed in our laboratory. Lactate levels were monitored during incremental cycling exercise challenges as a tool for hyperlactatemia. Six healthy individuals 18-45 y/o (4 males, 2 females) participated in the study. CLM devices were inserted subcutaneously in the postero-lateral trunk below the renal angle, one hour before the exercise challenge. Each exercise challenge consisted of a 3 to 12-min warm up period, followed by up to 7, 4-min incremental workload bouts separated by rest intervals. Continuous lactate measurements obtained from CLM were compared with commercial lactate analyzer (Abbott iSTAT) measurements of venous blood (plasma) drawn from the antecubital vein. Blood was drawn at up to 25 time points spanning the duration of before exercise, during exercise, and up to 120 min post exercise. Area under the curve (AUC), and delay time were calculated to compare the CLM readings with plasma lactate concentration. Average plasma lactate concentration increased from 1.02 to 16.21 mM. Ratio of AUC derived from CLM to plasma lactate was 1.025 (0.990-1.058). Average dynamic delay time of CLM to venous plasma lactate was 5.22 min (2.87-10.35). Insertion sites examined 48 h after CLM removal did not show signs of side effects and none required medical attention upon examination. The newly developed CLM has shown to be a promising tool to continuously measure lactate concentration in a minimally invasive fashion. Results indicate the CLM can provide needed trends in lactate over time. Such a device may be used in the future to improve treatment in clinical conditions such as sepsis.

Single-shot interferometric measurement of cavitation bubble dynamics.

We demonstrate an interferometric method to provide direct, single-shot measurements of cavitation bubble dynamics with nanoscale spatial and temporal resolution with results that closely match theoretical predictions. Implementation of this method reduces the need for expensive and complex ultra-high speed camera systems for the measurement of single cavitation events. This method can capture dynamics over large time intervals with sub-nanosecond temporal resolution and spatial precision surpassing the optical diffraction limit. We expect this method to have broad utility for examination of cavitation bubble dynamics, as well as for metrology applications such as optorheological materials characterization. This method provides an accurate approach for precise measurement of cavitation bubble dynamics suitable for metrology applications such as optorheological materials characterization.

Photostable and Proteolysis-Resistant Förster Resonance Energy Transfer-Based Calcium Biosensor.

Molecular sensors from protein engineering offer new methods to sensitively bind to and detect target analytes for a wide range of applications. For example, these sensors can be integrated into probes for implantation, and then yield new and valuable physiological information. Here, a new Förster resonance energy transfer (FRET)-based sensor is integrated with an optical fiber to yield a device measuring free Ca2+. This membrane encapsulated optical fiber (MEOF) device is composed of a sensor matrix that fills poly(tetrafluoroethylene) (PTFE) with an engineered troponin C (TnC) protein fused to a pair of FRET fluorophores. The FRET efficiency is modulated upon Ca2+ ion binding. The probe further comprises a second, size-excluding filter membrane that is synthesized by filling the pores of a PTFE matrix with a poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogel; this design ensures protection from circulating proteases and the foreign body response. The two membranes are stacked and placed on a thin, silica optical fiber for optical excitation and detection. Results show the biosensor responds to changes in Ca2+ concentration within minutes with a sensitivity ranging from 0.01 to 10 mM Ca2+, allowing discrimination of hyper- and hypocalcemia. Furthermore, the system reversibly binds Ca2+ to allow continuous monitoring. This work paves the way for the use of engineered structure-switching proteins for continuous optical monitoring in a large number of applications.

A pilot clinical trial of a near-infrared laser vaccine adjuvant: safety, tolerability, and cutaneous immune cell trafficking.

Many vaccines require adjuvants to enhance immunogenicity, but there are few safe and effective intradermal (i.d.) adjuvants. Murine studies have validated the potency of laser illumination of skin as an adjuvant for i.d. vaccination with advantages over traditional adjuvants. We report a pilot clinical trial of low-power, continuous-wave, near-infrared laser adjuvant treatment, representing the first human trial of the safety, tolerability, and cutaneous immune cell trafficking changes produced by the laser adjuvant. In this trial we demonstrated a maximum tolerable energy dose of 300 J/cm2 to a spot on the lower back. The irradiated spot was biopsied 4 h later, as was a control spot. Paired biopsies were submitted for histomorphologic and immunohistochemical evaluation in a blinded fashion as well as quantitative PCR analysis for chemokines and cytokines. Similar to prior murine studies, highly significant reductions in CD1a+ Langerhans cells in the dermis and CD11c+ dermal dendritic cells were observed, corresponding to the increased migratory activity of these cells; changes in the epidermis were not significant. There was no evidence of skin damage. The laser adjuvant is a safe, well-tolerated adjuvant for i.d. vaccination in humans and results in significant cutaneous immune cell trafficking.-Gelfand, J. A., Nazarian, R. M., Kashiwagi, S., Brauns, T., Martin, B., Kimizuka, Y., Korek, S., Botvinick, E., Elkins, K., Thomas, L., Locascio, J., Parry, B., Kelly, K. M., Poznansky, M. C. A pilot clinical trial of a near-infrared laser vaccine adjuvant: safety, tolerability, and cutaneous immune cell trafficking.

Evolution of Multivalent Nanoparticle Adhesion via Specific Molecular Interactions.

The targeted delivery of nanoparticle carriers holds tremendous potential to transform the detection and treatment of diseases. A major attribute of nanoparticles is the ability to form multiple bonds with target cells, which greatly improves the adhesion strength. However, the multivalent binding of nanoparticles is still poorly understood, particularly from a dynamic perspective. In previous experimental work, we studied the kinetics of nanoparticle adhesion and found that the rate of detachment decreased over time. Here, we have applied the adhesive dynamics simulation framework to investigate binding dynamics between an antibody-conjugated, 200-nm-diameter sphere and an ICAM-1-coated surface on the scale of individual bonds. We found that nano adhesive dynamics (NAD) simulations could replicate the time-varying nanoparticle detachment behavior that we observed in experiments. As expected, this behavior correlated with a steady increase in mean bond number with time, but this was attributed to bond accumulation only during the first second that nanoparticles were bound. Longer-term increases in bond number instead were manifested from nanoparticle detachment serving as a selection mechanism to eliminate nanoparticles that had randomly been confined to lower bond valencies. Thus, time-dependent nanoparticle detachment reflects an evolution of the remaining nanoparticle population toward higher overall bond valency. We also found that NAD simulations precisely matched experiments whenever mechanical force loads on bonds were high enough to directly induce rupture. These mechanical forces were in excess of 300 pN and primarily arose from the Brownian motion of the nanoparticle, but we also identified a valency-dependent contribution from bonds pulling on each other. In summary, we have achieved excellent kinetic consistency between NAD simulations and experiments, which has revealed new insights into the dynamics and biophysics of multivalent nanoparticle adhesion. In future work, we will leverage the simulation as a design tool for optimizing targeted nanoparticle agents.

Adenosine A(1) and prostaglandin E receptor 3 receptors mediate global airway contraction after local epithelial injury.

Epithelial injury and airway hyperresponsiveness are prominent features of asthma. We have previously demonstrated that laser ablation of single epithelial cells immediately induces global airway constriction through Ca(2+)-dependent smooth muscle shortening. The response is mediated by soluble mediators released from wounded single epithelial cells; however, the soluble mediators and signaling mechanisms have not been identified. In this study, we investigated the nature of the epithelial-derived soluble mediators and the associated signaling pathways that lead to the L-type voltage-dependent Ca(2+) channel (VGCC)-mediated Ca(2+) influx. We found that inhibition of adenosine A1 receptors (or removal of adenosine with adenosine deaminase), cyclooxygenase (COX)-2 or prostaglandin E receptor 3 (EP3) receptors, epidermal growth factor receptor (EGFR), or platelet-derived growth factor receptor (PDGFR) all significantly blocked Ca(2+) oscillations in smooth muscle cells and airway contraction induced by local epithelial injury. Using selective agonists to activate the receptors in the presence and absence of selective receptor antagonists, we found that adenosine activated the signaling pathway A1R→EGFR/PDGFR→COX-2→EP3→VGCCs→calcium-induced calcium release, leading to intracellular Ca(2+) oscillations in airway smooth muscle cells and airway constriction.

Effect of Porous Substrate Topographies on Cell Dynamics: A Computational Study.

Controlling cell-substrate interactions via the microstructural characteristics of biomaterials offers an advantageous path for modulating cell dynamics, mechanosensing, and migration, as well as for designing immune-modulating implants, all without the drawbacks of chemical-based triggers. Specifically, recent in vivo studies have suggested that a porous implant's microscale curvature landscape can significantly impact cell behavior and ultimately the immune response. To investigate such cell-substrate interactions, we utilized a 3D computational model incorporating the minimum necessary physics of cell migration and cell-substrate interactions needed to replicate known in vitro behaviors. This model specifically incorporates the effect of membrane tension, which was found to be necessary to replicate in vitro cell behavior on curved surfaces. Our simulated substrates represent two classes of porous materials recently used in implant studies, which have markedly different microscale curvature distributions and pore geometries. We found distinct differences between the overall migration behaviors, shapes, and actin polymerization dynamics of cells interacting with the two substrates. These differences were correlated to the shape energy of the cells as they interacted with the porous substrates, in effect interpreting substrate topography as an energetic landscape interrogated by cells. Our results demonstrate that microscale curvature directly influences cell shape and migration and, therefore, is likely to influence cell behavior. This supports further investigation of the relationship between the surface topography of implanted materials and the characteristic immune response, a complete understanding of which would broadly advance principles of biomaterial design.

Patterned photocrosslinking to establish stiffness anisotropies in fibrous 3D hydrogels.

Cells are known to constantly interact with their local extracellular matrix (ECM) and respond to a variety of biochemical and mechanical cues received from the ECM. Nonetheless, comprehensive understanding of cell-ECM interactions has been elusive. Many studies rely on analysis of cell behavior on 2D substrates, which do not reflect a natural cell environment. Further, lack of dynamic control over local stiffness anisotropies and fiber alignment hinders progress in studies in naturally derived fibrous 3D cultures. Here, we present a cell-safe method of patterned photocrosslinking, which can aid in studying biological hypotheses related to mechanotransduction in 3D hydrogels. As previously described by our group, ruthenium-catalyzed photocrosslinking (RCP) of selected ECM regions promotes localized increase in stiffness mediated by focused blue laser light in a confocal microscope. In this study, we further demonstrate that RCP can induce localized strain stiffening and fiber alignment outside of the selected crosslinked region and induce stiffness anisotropy biased towards the direction of fiber alignment. MDA-MB-231 cells are shown to respond to RCP-induced changes in local ECM architecture and display directional bias towards the direction of fiber alignment, as compared to control cells. Further, the effect of patterned crosslinking on a stiffness landscape is measured using multi-axes optical tweezers active microrheology (AMR) with backscattered laser beam illumination. AMR validates RCP as a suitable tool for creating distinct stiffness anisotropies which promote directed migration of cells, further underscoring the usefulness of RCP in cell-ECM studies. STATEMENT OF SIGNIFICANCE: Studies on cell-ECM interactions in 3D cultures have often been hindered by the lack of available tools to dynamically alter local ECM stiffness and fiber alignment. Here, we present a non-invasive, cell-safe and easily applicable method of patterned photocrosslinking, which can aid in studying biological hypotheses in fibrous 3D hydrogels. Ruthenium-catalyzed crosslinking (RCP) of selected fibrin ECM regions promotes localized increase in stiffness and creates distinct stiffness anisotropies in the presence of the focused blue laser light. Outside of the crosslinked region, RCP causes fiber alignment and strain stiffening in the ECM, verified using multi-axes optical tweezers active microrheology (AMR). Following RCP, human breast cancer MDA-MB-231 exhibit directed cell migration, validating usefulness of this method in cell-ECM studies.

Cell mediated remodeling of stiffness matched collagen and fibrin scaffolds.

Cells are known to continuously remodel their local extracellular matrix (ECM) and in a reciprocal way, they can also respond to mechanical and biochemical properties of their fibrous environment. In this study, we measured how stiffness around dermal fibroblasts (DFs) and human fibrosarcoma HT1080 cells differs with concentration of rat tail type 1 collagen (T1C) and type of ECM. Peri-cellular stiffness was probed in four directions using multi-axes optical tweezers active microrheology (AMR). First, we found that neither cell type significantly altered local stiffness landscape at different concentrations of T1C. Next, rat tail T1C, bovine skin T1C and fibrin cell-free hydrogels were polymerized at concentrations formulated to match median stiffness value. Each of these hydrogels exhibited distinct fiber architecture. Stiffness landscape and fibronectin secretion, but not nuclear/cytoplasmic YAP ratio differed with ECM type. Further, cell response to Y27632 or BB94 treatments, inhibiting cell contractility and activity of matrix metalloproteinases, respectively, was also dependent on ECM type. Given differential effect of tested ECMs on peri-cellular stiffness landscape, treatment effect and cell properties, this study underscores the need for peri-cellular and not bulk stiffness measurements in studies on cellular mechanotransduction.

Transcutaneous Flexible Sensor for In Vivo Photonic Detection of pH and Lactate.

Clinical research shows that frequent measurements of both pH and lactate can help guide therapy and improve patient outcome. However, current methods of sampling blood pH and lactate make it impractical to take readings frequently (due to the heightened risk of blood infection and anemia). As a solution, we have engineered a subcutaneous pH and lactate sensor (PALS) that can provide continuous, physiologically relevant measurements. To measure pH, a sheet containing a pH-sensitive fluorescent dye is placed over 400 and 465 nm light-emitting diodes (LEDs) and a filter-coated photodetector. The filter-coated photodetector collects an emitted signal from the dye for each LED excitation, and the ratio of the emitted signals is used to monitor pH. To measure lactate, two sensing sheets comprising an oxygen-sensitive phosphorescent dye are each mounted to a 625 nm LED. One sheet additionally comprises the enzyme lactate oxidase. The LEDs are sequentially modulated to excite the sensing sheets, and their phase shift at the LED drive frequency is used to monitor lactate. In vitro results indicate that PALS successfully records pH changes from 6.92 to 7.70, allowing for discrimination between acidosis and alkalosis, and can track lactate levels up to 9 mM. Both sensing strategies exhibit fast rise times (< 5 min) and stable measurements. Multianalyte in vitro models of physiological disorders show that the sensor measurements consistently quantify the expected pathophysiological trends without cross talk; in vivo rabbit testing further indicates usefulness in the clinical setting.

Micropyramid-patterned, oxygen-permeable bottomed dish for high density culture of pancreatic islets.

The need for maintaining cell-spheroid viability and function within high-density cultures is unmet for various clinical and experimental applications, including cell therapies. One immediate application is for transplantation of pancreatic islets, a clinically recognized treatment option to cure type 1 diabetes; islets are isolated from a donor for subsequent culture prior to transplantation. However, high seeding conditions cause unsolicited fusion of multiple spheroids, thereby limiting oxygen diffusion to induce hypoxic cell death. Here we introduce a culture dish incorporating a micropyramid-patterned surface to prevent the unsolicited fusion and oxygen-permeable bottom for optimal oxygen environment. A 400µm-thick, oxygen-permeable polydimethylsiloxane sheet topped with micropyramid pattern of 400µm-base and 200µm-height was fabricated to apply to the 24-well plate format. The micropyramid pattern separated the individual pancreatic islets to prevent the fusion of multiple islets. This platform supported the high oxygen demand of islets at high seeding density at 260 islet equivalents cm-2, a 2-3-fold higher seeding density compared to the conventional islet culture used in a preparation for the clinical islet transplantations, demonstrating improved islet morphology, metabolism and function in a 4 d-culture. Transplantation of these islets into immunodeficient diabetic mice exhibited significantly improved engraftment to achieve euglycemia compared to islets cultured in the conventional culture wells. Collectively, this simple design modification allows for high-density cultures of three-dimensional cell spheroids to improve the viability and function for an array of investigational and clinical replacement tissues.

Visualizing the mechanical activation of Src.

The mechanical environment crucially influences many cell functions. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin-cytoskeleton interaction, which is essential for the transduction of mechanical stimuli. Using fluorescent resonance energy transfer (FRET), here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells (HUVECs) by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed (mean +/- s.e.m.) of 18.1 +/- 1.7 nm s(-1). This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations.

Dermal fibroblasts and triple-negative mammary epithelial cancer cells differentially stiffen their local matrix.

The bulk measurement of extracellular matrix (ECM) stiffness is commonly used in mechanobiology. However, past studies by our group show that peri-cellular stiffness is quite heterogeneous and divergent from the bulk. We use optical tweezers active microrheology (AMR) to quantify how two phenotypically distinct migratory cell lines establish dissimilar patterns of peri-cellular stiffness. Dermal fibroblasts (DFs) and triple-negative human breast cancer cells MDA-MB-231 (MDAs) were embedded within type 1 collagen (T1C) hydrogels polymerized at two concentrations: 1.0 mg/ml and 1.5 mg/ml. We found DFs increase the local stiffness of 1.0 mg/ml T1C hydrogels but, surprisingly, do not alter the stiffness of 1.5 mg/ml T1C hydrogels. In contrast, MDAs predominantly do not stiffen T1C hydrogels as compared to cell-free controls. The results suggest that MDAs adapt to the bulk ECM stiffness, while DFs regulate local stiffness to levels they intrinsically prefer. In other experiments, cells were treated with transforming growth factor-ß1 (TGF-ß1), glucose, or ROCK inhibitor Y27632, which have known effects on DFs and MDAs related to migration, proliferation, and contractility. The results show that TGF-ß1 alters stiffness anisotropy, while glucose increases stiffness magnitude around DFs but not MDAs and Y27632 treatment inhibits cell-mediated stiffening. Both cell lines exhibit an elongated morphology and local stiffness anisotropy, where the stiffer axis depends on the cell line, T1C concentration, and treatment. In summary, our findings demonstrate that AMR reveals otherwise masked mechanical properties such as spatial gradients and anisotropy, which are known to affect cell behavior at the macro-scale. The same properties manifest with similar magnitude around single cells.

Non-Invasive Monitoring of Oxygen Tension and Oxygen Transport Inside Subcutaneous Devices After H2S Treatment.

Medical devices for cell therapy can be improved through prevascularization. In this work we study the vascularization of a porous polymer device, previously used by our group for pancreatic islet transplantation with results indicating improved glycemic control. Oxygen partial pressure within such devices was monitored non-invasively using an optical technique. Oxygen-sensitive tubes were fabricated and placed inside devices prior to subcutaneous implantation in nude mice. We tested the hypothesis that vascularization will be enhanced by administration of the pro-angiogenic factor hydrogen sulfide (H2S). We found that oxygen dynamics were unique to each implant and that the administration of H2S does not result in significant changes in perfusion of the devices as compared with control. These observations suggest that vascular perfusion and density are not necessarily correlated, and that the rate of vascularization was not enhanced by the pro-angiogenic agent.

Laser cavitation rheology for measurement of elastic moduli and failure strain within hydrogels.

We introduce laser cavitation rheology (LCR) as a minimally-invasive optical method to characterize mechanical properties within the interior of biological and synthetic aqueous soft materials at high strain-rates. We utilized time-resolved photography to measure cavitation bubble dynamics generated by the delivery of focused 500 ps duration laser radiation at λ = 532 nm within fibrin hydrogels at pulse energies of Ep = 12, 18 µJ and within polyethylene glycol (600) diacrylate (PEG (600) DA) hydrogels at Ep = 2, 5, 12 µJ. Elastic moduli and failure strains of fibrin and PEG (600) DA hydrogels were calculated from these measurements by determining parameter values which provide the best fit of the measured data to a theoretical model of cavitation bubble dynamics in a Neo-Hookean viscoelastic medium subject to material failure. We demonstrate the use of this method to retrieve the local, interior elastic modulus of these hydrogels and both the radial and circumferential failure strains.

A bench-top model of middle ear effusion diagnosed with optical tympanometry.

OBJECTIVES: To assess the validity of a bench-top model of an optical tympanometry device to diagnose in vitro model of middle ear effusion (MEE). METHODS AND MATERIALS: We illuminated an in vitro model of ear canal and tympanic membrane with broadband light and relayed remitted light to a spectrometer system. We then used our proprietary algorithm to extract spectral features that, together with our logistic regression classifiers, led us to calculate a set of simplified indices related to different middle ear states. Our model included a glass vial covered with a porcine submucosa (representing the tympanic membrane) and filled with air, water, or milk solution (representing different MEE), and a set of cover-glass slips filled with either blood (representing erythema) or cerumen. By interchanging fluid types and cover-glass slips, we made measurements on combinations corresponding to normal healthy ear and purulent or serous MEE. RESULTS: Each simulated condition had a distinct spectral profile, which was then employed by our algorithm to discriminate clean and cerumen-covered purulent and serous MEE. Two logistic purulent and serous MEE classifiers correctly classified all in vitro middle ear states with 100% accuracy assessed by leave-one-out and k-fold cross validation. CONCLUSIONS: This proof-of-concept in vitro study addressed an unmet need by introducing a device that easily and accurately can assess middle ear effusion. Future in vivo studies aimed at collecting data from clinical settings are warranted to further elucidate the validity of the technology in diagnosing pediatric acute otitis media.

The Effect of a Fast-Releasing Hydrogen Sulfide Donor on Vascularization of Subcutaneous Scaffolds in Immunocompetent and Immunocompromised Mice.

Islet transplantation into subcutaneous polymer scaffolds has shown to successfully induce normoglycemia in type 1 diabetes models. Vascularization of these scaffolds is imperative for optimal control of glucose levels. We studied the effect of the vascular stimulator hydrogen sulfide (H2S) on the degree of vascularization of a scaffold and the role of the immune system in this process. Scaffolds were subcutaneously implanted in immunocompetent C57BL/6 and immunocompromised nude mice. Mice received twice-daily intraperitoneal injections of the fast-releasing H2S donor sodium hydrosulfide (NaHS, 25 or 50 µmol/kg) or saline for 28 days. After 63 days the vascular network was analyzed by histology and gene expression. Here we showed that the vascularization of a subcutaneous scaffold in nude mice was significantly impaired by H2S treatment. Both the CD31 gene and protein expression were reduced in these scaffolds compared to the saline-treated controls. In C57BL/6 mice, the opposite was found, the vascularization of the scaffold was significantly increased by H2S. The mRNA expression of the angiogenesis marker CD105 was significantly increased compared to the controls as well as the number of CD31 positive blood vessels. In conclusion, the immune system plays an important role in the H2S mediated effect on vascularization of subcutaneous scaffolds.

Selective stiffening of fibrin hydrogels with micron resolution via photocrosslinking.

Fibrin hydrogels are used as a model system for studying cell-ECM biophysical interactions. Bulk mechanical stiffness of these hydrogels has been correlated to mechanotransduction and downstream signaling. However, stiffness values proximal to cells can vary by orders of magnitude at the length scale of microns. Patterning of matrix stiffness at this spatial scale can be useful in studying such interactions. Here we present and evaluate a technique to selectively stiffen defined regions within a fibrin hydrogel. Laser scanning illumination activates ruthenium-catalyzed crosslinking of fibrin tyrosine residues, resulting in tunable stiffness changes spanning distances as small as a few microns and a localized compaction of the material. As probed by active microrheology, stiffness increases by as much as 25X, similar to previously observed stiffness changes around single cells in 3D culture. In summary, our method allows for selective modification of fibrin stiffness at the micron scale with the potential to create complex patterns, which could be valuable for the investigation of mechanotransduction in a biologically meaningful way. STATEMENT OF SIGNIFICANCE: Fibrin hydrogels are used as a naturally derived model to study interactions between cells and their surrounding extracellular matrix (ECM). ECM stiffness influences cell state. Cells in 3D culture considerably modify the stiffness of their pericellular space, which can be quite heterogeneous at the micron-scale. Here we present and evaluate a method to pattern stiffness within fibrin hydrogels using a laser scanning confocal microscope and selective photo crosslinking. We believe that this technique can aid future studies of cell-ECM interactions by enabling researchers to modify the pericellular distribution of stiffness.