Effect of sleep on overnight cerebrospinal fluid amyloid ß kinetics.

Sleep disturbances are associated with future risk of Alzheimer disease. Disrupted sleep increases soluble amyloid ß, suggesting a mechanism for sleep disturbances to increase Alzheimer disease risk. We tested this response in humans using indwelling lumbar catheters to serially sample cerebrospinal fluid while participants were sleep-deprived, treated with sodium oxybate, or allowed to sleep normally. All participants were infused with 13 C6 -leucine to measure amyloid ß kinetics. We found that sleep deprivation increased overnight amyloid ß38, amyloid ß40, and amyloid ß42 levels by 25 to 30% via increased overnight amyloid ß production relative to sleeping controls. These findings suggest that disrupted sleep increases Alzheimer disease risk via increased amyloid ß production. Ann Neurol 2018;83:197-204.

Age and amyloid effects on human central nervous system amyloid-beta kinetics.

OBJECTIVE: Age is the single greatest risk factor for Alzheimer's disease (AD), with the incidence doubling every 5 years after age 65. However, our understanding of the mechanistic relationship between increasing age and the risk for AD is currently limited. We therefore sought to determine the relationship between age, amyloidosis, and amyloid-beta (Aß) kinetics in the central nervous system (CNS) of humans. METHODS: Aß kinetics were analyzed in 112 participants and compared to the ages of participants and the amount of amyloid deposition. RESULTS: We found a highly significant correlation between increasing age and slowed Aß turnover rates (2.5-fold longer half-life over five decades of age). In addition, we found independent effects on Aß42 kinetics specifically in participants with amyloid deposition. Amyloidosis was associated with a higher (>50%) irreversible loss of soluble Aß42 and a 10-fold higher Aß42 reversible exchange rate. INTERPRETATION: These findings reveal a mechanistic link between human aging and the risk of amyloidosis, which may be owing to a dramatic slowing of Aß turnover, increasing the likelihood of protein misfolding that leads to deposition. Alterations in Aß kinetics associated with aging and amyloidosis suggest opportunities for diagnostic and therapeutic strategies. More generally, this study provides an example of how changes in protein turnover kinetics can be used to detect physiological and pathophysiological changes and may be applicable to other proteinopathies.

Amyloid-ß efflux from the central nervous system into the plasma.

OBJECTIVE: The aim of this study was to measure the flux of amyloid-ß (Aß) across the human cerebral capillary bed to determine whether transport into the blood is a significant mechanism of clearance for Aß produced in the central nervous system (CNS). METHODS: Time-matched blood samples were simultaneously collected from a cerebral vein (including the sigmoid sinus, inferior petrosal sinus, and the internal jugular vein), femoral vein, and radial artery of patients undergoing inferior petrosal sinus sampling. For each plasma sample, Aß concentration was assessed by 3 assays, and the venous to arterial Aß concentration ratios were determined. RESULTS: Aß concentration was increased by ∼7.5% in venous blood leaving the CNS capillary bed compared to arterial blood, indicating efflux from the CNS into the peripheral blood (p < 0.0001). There was no difference in peripheral venous Aß concentration compared to arterial blood concentration. INTERPRETATION: Our results are consistent with clearance of CNS-derived Aß into the venous blood supply with no increase from a peripheral capillary bed. Modeling these results suggests that direct transport of Aß across the blood-brain barrier accounts for ∼25% of Aß clearance, and reabsorption of cerebrospinal fluid Aß accounts for ∼25% of the total CNS Aß clearance in humans. Ann Neurol 2014;76:837-844.

Ultralow protein adsorbing coatings from clickable PEG nanogel solutions: benefits of attachment under salt-induced phase separation conditions and comparison with PEG/albumin nanogel coatings.

Clickable nanogel solutions were synthesized by using the copper catalyzed azide/alkyne cycloaddition (CuAAC) to partially polymerize solutions of azide and alkyne functionalized poly(ethylene glycol) (PEG) monomers. Coatings were fabricated using a second click reaction: a UV thiol-yne attachment of the nanogel solutions to mercaptosilanated glass. Because the CuAAC reaction was effectively halted by the addition of a copper-chelator, we were able to prevent bulk gelation and limit the coating thickness to a single monolayer of nanogels in the absence of the solution reaction. This enabled the inclusion of kosmotropic salts, which caused the PEG to phase-separate and nearly double the nanogel packing density, as confirmed by quartz crystal microbalance with dissipation (QCM-D). Protein adsorption was analyzed by single molecule counting with total internal reflection fluorescence (TIRF) microscopy and cell adhesion assays. Coatings formed from the phase-separated clickable nanogel solutions attached with salt adsorbed significantly less fibrinogen than other 100% PEG coatings tested, as well as poly(L-lysine)-g-PEG (PLL-g-PEG) coatings. However, PEG/albumin nanogel coatings still outperformed the best 100% PEG clickable nanogel coatings. Additional surface cross-linking of the clickable nanogel coating in the presence of copper further reduced levels of fibrinogen adsorption closer to those of PEG/albumin nanogel coatings. However, this step negatively impacted long-term resistance to cell adhesion and dramatically altered the morphology of the coating by atomic force microscopy (AFM). The main benefit of the click strategy is that the partially polymerized solutions are stable almost indefinitely, allowing attachment in the phase-separated state without danger of bulk gelation, and thus producing the best performing 100% PEG coating that we have studied to date.

Importance of CSF-based Aß clearance with age in humans increases with declining efficacy of blood-brain barrier/proteolytic pathways.

The kinetics of amyloid beta turnover within human brain is still poorly understood. We previously found a dramatic decline in the turnover of Aß peptides in normal aging. It was not known if brain interstitial fluid/cerebrospinal fluid (ISF/CSF) fluid exchange, CSF turnover, blood-brain barrier function or proteolysis were affected by aging or the presence of ß amyloid plaques. Here, we describe a non-steady state physiological model developed to decouple CSF fluid transport from other processes. Kinetic parameters were estimated using: (1) MRI-derived brain volumes, (2) stable isotope labeling kinetics (SILK) of amyloid-ß peptide (Aß), and (3) lumbar CSF Aß concentration during SILK. Here we show that changes in blood-brain barrier transport and/or proteolysis were largely responsible for the age-related decline in Aß turnover rates. CSF-based clearance declined modestly in normal aging but became increasingly important due to the slowing of other processes. The magnitude of CSF-based clearance was also lower than that due to blood-brain barrier function plus proteolysis. These results suggest important roles for blood-brain barrier transport and proteolytic degradation of Aß in the development Alzheimer's Disease in humans.

Poly(ethylene glycol) microparticles produced by precipitation polymerization in aqueous solution.

Methods were developed to perform precipitation photopolymerization of PEG-diacrylate. Previously, comonomers have been added to PEG when precipitation polymerization was desired. In the present method, the LCST of the PEG itself was lowered by the addition of the kosmotropic salt sodium sulfate to an aqueous solution. Typical of a precipitation polymerization, small microparticles or microspheres (1-5 µm) resulted with relatively low polydispersity. However, aggregate formation was often severe, presumably because of a lack of stabilization of the phase-separated colloids. Microparticles were also produced by copoymerization of PEG-diacrylate with acrylic acid or aminoethylmethacrylate. The comonomers affected the zeta potential of the formed microparticles but not the size. The carboxyl groups of acrylic-acid-containing PEG microparticles were activated, and scaffolds were formed by mixing with amine-containing PEG microparticles. Although the scaffolds were relatively weak, human hepatoma cells showed excellent viability when present during microparticle cross-linking.

SILK studies - capturing the turnover of proteins linked to neurodegenerative diseases.

Alzheimer disease (AD) is one of several neurodegenerative diseases characterized by dysregulation, misfolding and accumulation of specific proteins in the CNS. The stable isotope labelling kinetics (SILK) technique is based on generating amino acids labelled with naturally occurring stable (that is, nonradioactive) isotopes of carbon and/or nitrogen. These labelled amino acids can then be incorporated into proteins, enabling rates of protein production and clearance to be determined in vivo and in vitro without the use of radioactive or chemical labels. Over the past decade, SILK studies have been used to determine the turnover of key pathogenic proteins amyloid-ß (Aß), tau and superoxide dismutase 1 (SOD1) in the cerebrospinal fluid of healthy individuals, patients with AD and those with other neurodegenerative diseases. These studies led to the identification of several factors that alter the production and/or clearance of these proteins, including age, sleep and disease-causing genetic mutations. SILK studies have also been used to measure Aß turnover in blood and within brain tissue. SILK studies offer the potential to elucidate the mechanisms underlying various neurodegenerative disease mechanisms, including neuroinflammation and synaptic dysfunction, and to demonstrate target engagement of novel disease-modifying therapies.

Endothelial cell migration on RGD-peptide-containing PEG hydrogels in the presence of sphingosine 1-phosphate.

Sphingosine 1-phosphate (S1P) is a potent chemokinetic agent for endothelial cells that is released by activated platelets. We previously developed Arg-Gly-Asp (RGD)-containing polyethylene glycol biomaterials for the controlled delivery of S1P to promote endothelialization. Here, we studied the effects of cell adhesion strength on S1P-stimulated endothelial cell migration in the presence of arterial levels of fluid shear stress, since an upward shift in optimal cell adhesion strengths may be beneficial for promoting long-term cell adhesion to materials. Two RGD peptides with different integrin-binding specificities were added to the polyethylene glycol hydrogels. A linear RGD bound primarily to beta(3) integrins, whereas a cyclic RGD bound through both beta(1) and beta(3) integrins. We observed increased focal adhesion formation and better long-term adhesion in flow with endothelial cells on linear RGD peptide, versus cyclic RGD, even though initial adhesion strengths were higher for cells on cyclic RGD. Addition of 100 nM S1P increased cell speed and random motility coefficients on both RGD peptides, with the largest increases found on cyclic RGD. For both peptides, much of the increase in cell migration speed was found for smaller cells (<1522 microm(2) projected area), although the large increases on cyclic RGD were also due to medium-sized cells (2288-3519 microm(2)). Overall, a compromise between high cell migration rates and long-term adhesion will be important in the design of materials that endothelialize after implantation.

Mass spectrometric mapping of fibrinogen conformations at poly(ethylene terephthalate) interfaces.

We have characterized the adsorption of bovine fibrinogen onto the biomedical polymer polyethylene terephthalate (PET) by performing mass spectrometric mapping with a lysine-reactive biotin label. After digestion with trypsin, MALDI-TOF mass spectrometry was used to detect peptides from biotinylated bovine fibrinogen, with the goal of identifying lysines that were more accessible for reaction with the chemical label after adsorption. Peptides within domains that are believed to contribute to heparin binding, leukocyte activation, and platelet adhesion were found to be biotin labeled only after bovine fibrinogen adsorbed to the PET surface. Additionally, the accessibility of lysine residues throughout the entire molecule was observed to increase as the concentration of the adsorbing bovine fibrinogen solution decreased, suggesting that the proximity of biologically active motifs to hydrophilic residues leads to their exposure. The surface area per adsorbed bovine fibrinogen molecule was quantified on PET using optical waveguide lightmode spectroscopy (OWLS), which revealed higher surface densities for bovine fibrinogen adsorbed from higher concentration solutions. By measuring changes in both the identity and conformation of proteins that adsorb from complex mixtures such as blood or plasma, this technique may have applications in fundamental studies of protein adsorption and may allow for more accurate predictions of the biocompatibility of materials.

Associations Between ß-Amyloid Kinetics and the ß-Amyloid Diurnal Pattern in the Central Nervous System.

Importance: Recent studies found that the concentration of amyloid-ß (Aß) fluctuates with the sleep-wake cycle. Although the amplitude of this day/night pattern attenuates with age and amyloid deposition, to our knowledge, the association of Aß kinetics (ie, production, turnover, and clearance) with this oscillation has not been studied. Objective: To determine the association between Aß kinetics, age, amyloid levels, and the Aß day/night pattern in humans. Design, Setting, and Participants: We measured Aß concentrations and kinetics in 77 adults aged 60 to 87 years with and without amyloid deposition by a novel precise mass spectrometry method at the Washington University School of Medicine in St Louis, Missouri. We compared findings of 2 orthogonal methods, enzyme-linked immunosorbent assay and mass spectrometry, to validate the day/night patterns and determine more precise estimates of the cosinor parameters. In vivo labeling of central nervous system proteins with stable isotopically labeled leucine was performed, and kinetics of Aß40 and Aß42 were measured. Interventions: Serial cerebrospinal fluid collection via indwelling lumbar catheter over 36 to 48 hours before, during, and after in vivo labeling, with a 9-hour primed constant infusion of 13C6-leucine. Main Outcomes and Measures: The amplitude, linear increase, and other cosinor measures of each participant's serial cerebrospinal fluid Aß concentrations and Aß turnover rates. Results: Of the 77 participants studied, 46 (59.7%) were men, and the mean (range) age was 72.6 (60.4-87.7) years. Day/night patterns in Aß concentrations were more sharply defined by the precise mass spectrometry method than by enzyme-linked immunosorbent assay (mean difference of SD of residuals: Aß40, -7.42 pM; P < .001; Aß42, -3.72 pM; P < .001). Amyloid deposition diminished day/night amplitude and linear increase of Aß42 but not of Aß40. Increased age diminished day/night amplitude of both Aß40 and Aß42. After controlling for amyloid deposition, amplitude of Aß40 was positively associated with production rates (r = 0.42; P < .001), while the linear rise was associated with turnover rates (r = 0.28; P < .05). The amplitude and linear rise of Aß42 were both associated with turnover (r = -0.38; P < .001) and production (r = 0.238; P < .05) rates. Conclusions and Relevance: Amyloid deposition is associated with premature loss of normal Aß42 day/night patterns in older adults, suggesting the previously reported effects of age and amyloid on Aß42 amplitude at least partially affect each other. Production and turnover rates suggest that day/night Aß patterns are modulated by both production and clearance mechanisms active in sleep-wake cycles and that amyloid deposition may impair normal circadian patterns. These findings may be important for the designs of future secondary prevention trials for Alzheimer disease.

Proteomic analysis of protein adsorption: serum amyloid P adsorbs to materials and promotes leukocyte adhesion.

Serum and plasma protein adsorption on materials was analyzed using gel electrophoresis and ion trap mass spectrometry. Following incubation of polypropylene, polyethylene terephthalate (PET), or polydimethylsiloxane (PDMS) with 5% serum for longer than 4 h, we found unexpectedly high amounts of the pentraxin serum amyloid P. It was previously shown that serum amyloid P is constitutively expressed in humans, functions as an opsonin, and interacts with the Fcgamma receptors on leukocytes. We demonstrate that serum amyloid P adsorbed to tissue culture polystyrene, PDMS, and PET promotes the adhesion of granulocytes and monocytes in the presence of calcium. The methods developed for these studies may be useful for the large-scale study of protein adsorption and do not rely on radiolabeling or the availability of antibodies.

Analysis of a compartmental model of amyloid beta production, irreversible loss and exchange in humans.

Amyloid beta (Aß) peptides, and in particular Aß42, are found in senile plaques associated with Alzheimer's disease. A compartmental model of Aß production, exchange and irreversible loss was recently developed to explain the kinetics of isotope-labeling of Aß peptides collected in cerebrospinal fluid (CSF) following infusion of stable isotope-labeled leucine in humans. The compartmental model allowed calculation of the rates of production, irreversible loss (or turnover) and short-term exchange of Aß peptides. Exchange of Aß42 was particularly pronounced in amyloid plaque-bearing participants. In the current work, we describe in much greater detail the characteristics of the compartmental model to two distinct audiences: physician-scientists and biokineticists. For physician-scientists, we describe through examples the types of questions the model can and cannot answer, as well as correct some misunderstandings of previous kinetic analyses applied to this type of isotope labeling data. For biokineticists, we perform a system identifiability analysis and a sensitivity analysis of the kinetic model to explore the global and local properties of the model. Combined, these analyses motivate simplifications from a more comprehensive physiological model to the final model that was previously presented. The analyses clearly demonstrate that the current dataset and compartmental model allow determination with confidence a single 'turnover' parameter, a single 'exchange' parameter and a single 'delay' parameter. When combined with CSF concentration data for the Aß peptides, production rates may also be obtained.

A modular, plasmin-sensitive, clickable poly(ethylene glycol)-heparin-laminin microsphere system for establishing growth factor gradients in nerve guidance conduits.

Peripheral nerve regeneration is a complex problem that, despite many advancements and innovations, still has sub-optimal outcomes. Compared to biologically derived acellular nerve grafts and autografts, completely synthetic nerve guidance conduits (NGC), which allow for precise engineering of their properties, are promising but still far from optimal. We have developed an almost entirely synthetic NGC that allows control of soluble growth factor delivery kinetics, cell-initiated degradability and cell attachment. We have focused on the spatial patterning of glial-cell derived human neurotrophic factor (GDNF), which promotes motor axon extension. The base scaffolds consisted of heparin-containing poly(ethylene glycol) (PEG) microspheres. The modular microsphere format greatly simplifies the formation of concentration gradients of reversibly bound GDNF. To facilitate axon extension, we engineered the microspheres with tunable plasmin degradability. 'Click' cross-linking chemistries were also added to allow scaffold formation without risk of covalently coupling the growth factor to the scaffold. Cell adhesion was promoted by covalently bound laminin. GDNF that was released from these microspheres was confirmed to retain its activity. Graded scaffolds were formed inside silicone conduits using 3D-printed holders. The fully formed NGC's contained plasmin-degradable PEG/heparin scaffolds that developed linear gradients in reversibly bound GDNF. The NGC's were implanted into rats with severed sciatic nerves to confirm in vivo degradability and lack of a major foreign body response. The NGC's also promoted robust axonal regeneration into the conduit.

The effect of the linker on the hydrolysis rate of drug-linked ester bonds.

Tailoring the length of a sulfide containing linker adjusts the hydrolysis of a drug-linked ester bond to values appropriate for once-a-week administrations. A model drug of paclitaxel was coupled using a hydrolyzable linker to a poly(ethylene glycol) macromonomer, via a conjugate addition reaction between a thiol and an acrylamide. The macromonomers were synthesized in three steps with an average overall yield of 70%. By changing the length of the linker from 3-sulfanylpropionyl to 4-sulfanylbutyryl, the half-life time of the release of the drug could be increased from 4.2+/-0.1 to 14.0+/-0.2 days. Drug-containing hydrogels were prepared by radical photopolymerization of these macromonomers with either the 3-sulfanylpropionyl or the 4-sulfanylbutyryl linker. The release of the drug from these hydrogels followed similar trends as the release of the drug from the soluble polymer-drug conjugates. The synthetic methodology employed does not involve the use of coupling reagents in the final conjugation between the drug and the polymer, excluding the presence of potential toxic residuals. The conjugation method is relatively simple and is applicable to nearly any hydroxyl-containing drugs.

Controlled release and gradient formation of human glial-cell derived neurotrophic factor from heparinated poly(ethylene glycol) microsphere-based scaffolds.

Introduction of spatial patterning of proteins, while retaining activity and releasability, is critical for the field of regenerative medicine. Reversible binding to heparin, which many biological molecules exhibit, is one potential pathway to achieve this goal. We have covalently bound heparin to poly(ethylene glycol) (PEG) microspheres to create useful spatial patterns of glial-cell derived human neurotrophic factor (GDNF) in scaffolds to promote peripheral nerve regeneration. Labeled GDNF was incubated with heparinated microspheres that were subsequently centrifuged into cylindrical scaffolds in distinct layers containing different concentrations of GDNF. The GDNF was then allowed to diffuse out of the scaffold, and release was tracked via fluorescent scanning confocal microscopy. The measured release profile was compared to predicted Fickian models. Solutions of reaction-diffusion equations suggested the concentrations of GDNF in each discrete layer that would result in a nearly linear concentration gradient over much of the length of the scaffold. The agreement between the predicted and measured GDNF concentration gradients was high. Multilayer scaffolds with different amounts of heparin and GDNF and different crosslinking densities allow the design of a wide variety of gradients and release kinetics. Additionally, fabrication is much simpler and more robust than typical gradient-forming systems due to the low viscosity of the microsphere solutions compared to gelating solutions, which can easily result in premature gelation or the trapping of air bubbles with a nerve guidance conduit. The microsphere-based method provides a framework for producing specific growth factor gradients in conduits designed to enhance nerve regeneration.

Clickable Poly(ethylene glycol)-Microsphere-Based Cell Scaffolds.

Clickable poly(ethylene glycol) (PEG) derivatives are used with two sequential aqueous two-phase systems to produce microsphere-based scaffolds for cell encapsulation. In the first step, sodium sulfate causes phase separation of the clickable PEG precursors and is followed by rapid geleation to form microspheres in the absence of organic solvent or surfactant. The microspheres are washed and then deswollen in dextran solutions in the presence of cells, producing tightly packed scaffolds that can be easily handled while also maintaining porosity. Endothelial cells included during microsphere scaffold formation show high viability. The clickable PEG-microsphere-based cell scaffolds open up new avenues for manipulating scaffold architecture as compared with simple bulk hydrogels.

Direct reprogramming of mouse fibroblasts to cardiomyocyte-like cells using Yamanaka factors on engineered poly(ethylene glycol) (PEG) hydrogels.

Direct reprogramming strategies enable rapid conversion of somatic cells to cardiomyocytes or cardiomyocyte-like cells without going through the pluripotent state. A recently described protocol couples Yamanaka factor induction with pluripotency inhibition followed by BMP4 treatment to achieve rapid reprogramming of mouse fibroblasts to beating cardiomyocyte-like cells. The original study was performed using Matrigel-coated tissue culture polystyrene (TCPS), a stiff material that also non-specifically adsorbs serum proteins. Protein adsorption-resistant poly(ethylene glycol) (PEG) materials can be covalently modified to present precise concentrations of adhesion proteins or peptides without the unintended effects of non-specifically adsorbed proteins. Here, we describe an improved protocol that incorporates custom-engineered materials. We first reproduced the Efe et al. protocol on Matrigel-coated TCPS (the original material), reprogramming adult mouse tail-tip mouse fibroblasts (TTF) and mouse embryonic fibroblasts (MEF) to cardiomyocyte-like cells that demonstrated striated sarcomeric α-actinin staining, spontaneous calcium transients, and visible beating. We then designed poly(ethylene glycol) culture substrates to promote MEF adhesion via laminin and RGD-binding integrins. PEG hydrogels improved proliferation and reprogramming efficiency (evidenced by beating patch number and area, gene expression, and flow cytometry), yielding almost twice the number of sarcomeric α-actinin positive cardiomyocyte-like cells as the originally described substrate. These results illustrate that cellular reprogramming may be enhanced using custom-engineered materials.

Increased in vivo amyloid-ß42 production, exchange, and loss in presenilin mutation carriers.

Alzheimer's disease (AD) is hypothesized to be caused by an overproduction or reduced clearance of amyloid-ß (Aß) peptide. Autosomal dominant AD (ADAD) caused by mutations in the presenilin (PSEN) gene have been postulated to result from increased production of Aß42 compared to Aß40 in the central nervous system (CNS). This has been demonstrated in rodent models of ADAD but not in human mutation carriers. We used compartmental modeling of stable isotope labeling kinetic (SILK) studies in human carriers of PSEN mutations and related noncarriers to evaluate the pathophysiological effects of PSEN1 and PSEN2 mutations on the production and turnover of Aß isoforms. We compared these findings by mutation status and amount of fibrillar amyloid deposition as measured by positron emission tomography (PET) using the amyloid tracer Pittsburgh compound B (PIB). CNS Aß42 to Aß40 production rates were 24% higher in mutation carriers compared to noncarriers, and this was independent of fibrillar amyloid deposits quantified by PET PIB imaging. The fractional turnover rate of soluble Aß42 relative to Aß40 was 65% faster in mutation carriers and correlated with amyloid deposition, consistent with increased deposition of Aß42 into plaques, leading to reduced recovery of Aß42 in cerebrospinal fluid (CSF). Reversible exchange of Aß42 peptides with preexisting unlabeled peptide was observed in the presence of plaques. These findings support the hypothesis that Aß42 is overproduced in the CNS of humans with PSEN mutations that cause AD, and demonstrate that soluble Aß42 turnover and exchange processes are altered in the presence of amyloid plaques, causing a reduction in Aß42 concentrations in the CSF.

Long-term culture of HL-1 cardiomyocytes in modular poly(ethylene glycol) microsphere-based scaffolds crosslinked in the phase-separated state.

Poly(ethylene glycol) (PEG) microspheres were assembled around HL-1 cardiomyocytes to produce highly porous modular scaffolds. In this study we took advantage of the immiscibility of PEG and dextran to improve upon our previously described modular scaffold fabrication methods. Phase separating the PEG microspheres in dextran solutions caused them to rapidly deswell and crosslink together, eliminating the need for serum protein-based crosslinking. This also led to a dramatic increase in the stiffness of the scaffolds and greatly improved the handling characteristics. HL-1 cardiomyocytes were present during microsphere crosslinking in the cytocompatible dextran solution, exhibiting high cell viability following scaffold formation. Over the course of 2 weeks a 9-fold expansion in cell number was observed. The cardiac functional markers sarcomeric α-actinin and connexin 43 were expressed at 13 and 24 days after scaffold formation. HL-1 cells were spontaneously depolarizing 38 days after scaffold formation, which was visualized by confocal microscopy using a calcium-sensitive dye. Electrical stimulation resulted in synchronization of activation peaks throughout the scaffolds. These findings demonstrate that PEG microsphere scaffolds fabricated in the presence of dextran can support the long-term three-dimensional culture of cells, suggesting applications in cardiovascular tissue engineering.