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The kinetics of amyloid beta turnover within human brain is still poorly understood. We previously found a dramatic decline in the turnover of Aβ 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 β 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-β peptide (Aβ), and (3) lumbar CSF Aβ 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 Aβ 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 Aβ in the development Alzheimer's Disease in humans., (© 2022. The Author(s).)

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-β (Aβ), 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 Aβ 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.

Sleep disturbances are associated with future risk of Alzheimer disease. Disrupted sleep increases soluble amyloid β, 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 C 6 -leucine to measure amyloid β kinetics. We found that sleep deprivation increased overnight amyloid β38, amyloid β40, and amyloid β42 levels by 25 to 30% via increased overnight amyloid β production relative to sleeping controls. These findings suggest that disrupted sleep increases Alzheimer disease risk via increased amyloid β production. Ann Neurol 2018;83:197-204., (© 2017 American Neurological Association.)

Importance: Recent studies found that the concentration of amyloid-β (Aβ) 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 Aβ kinetics (ie, production, turnover, and clearance) with this oscillation has not been studied., Objective: To determine the association between Aβ kinetics, age, amyloid levels, and the Aβ day/night pattern in humans., Design, Setting, and Participants: We measured Aβ 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 Aβ40 and Aβ42 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 Aβ concentrations and Aβ 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 Aβ concentrations were more sharply defined by the precise mass spectrometry method than by enzyme-linked immunosorbent assay (mean difference of SD of residuals: Aβ40, -7.42 pM; P < .001; Aβ42, -3.72 pM; P < .001). Amyloid deposition diminished day/night amplitude and linear increase of Aβ42 but not of Aβ40. Increased age diminished day/night amplitude of both Aβ40 and Aβ42. After controlling for amyloid deposition, amplitude of Aβ40 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 Aβ42 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 Aβ42 day/night patterns in older adults, suggesting the previously reported effects of age and amyloid on Aβ42 amplitude at least partially affect each other. Production and turnover rates suggest that day/night Aβ 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., Competing Interests: Dr. Elbert reports no conflicts.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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 (Aβ) kinetics in the central nervous system (CNS) of humans., Methods: Aβ 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 Aβ turnover rates (2.5-fold longer half-life over five decades of age). In addition, we found independent effects on Aβ42 kinetics specifically in participants with amyloid deposition. Amyloidosis was associated with a higher (>50%) irreversible loss of soluble Aβ42 and a 10-fold higher Aβ42 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 Aβ turnover, increasing the likelihood of protein misfolding that leads to deposition. Alterations in Aβ 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., (© 2015 American Neurological Association.)

Amyloid beta (Aβ) peptides, and in particular Aβ42, are found in senile plaques associated with Alzheimer's disease. A compartmental model of Aβ production, exchange and irreversible loss was recently developed to explain the kinetics of isotope-labeling of Aβ 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 Aβ peptides. Exchange of Aβ42 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 Aβ peptides, production rates may also be obtained., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to measure the flux of amyloid-β (Aβ) across the human cerebral capillary bed to determine whether transport into the blood is a significant mechanism of clearance for Aβ 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, Aβ concentration was assessed by 3 assays, and the venous to arterial Aβ concentration ratios were determined., Results: Aβ 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 Aβ concentration compared to arterial blood concentration., Interpretation: Our results are consistent with clearance of CNS-derived Aβ into the venous blood supply with no increase from a peripheral capillary bed. Modeling these results suggests that direct transport of Aβ across the blood-brain barrier accounts for ∼25% of Aβ clearance, and reabsorption of cerebrospinal fluid Aβ accounts for ∼25% of the total CNS Aβ clearance in humans. Ann Neurol 2014;76:837-844., (© 2014 American Neurological Association.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Alzheimer's disease (AD) is hypothesized to be caused by an overproduction or reduced clearance of amyloid-β (Aβ) peptide. Autosomal dominant AD (ADAD) caused by mutations in the presenilin (PSEN) gene have been postulated to result from increased production of Aβ42 compared to Aβ40 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 Aβ 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 Aβ42 to Aβ40 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 Aβ42 relative to Aβ40 was 65% faster in mutation carriers and correlated with amyloid deposition, consistent with increased deposition of Aβ42 into plaques, leading to reduced recovery of Aβ42 in cerebrospinal fluid (CSF). Reversible exchange of Aβ42 peptides with preexisting unlabeled peptide was observed in the presence of plaques. These findings support the hypothesis that Aβ42 is overproduced in the CNS of humans with PSEN mutations that cause AD, and demonstrate that soluble Aβ42 turnover and exchange processes are altered in the presence of amyloid plaques, causing a reduction in Aβ42 concentrations in the CSF.

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.

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.

Conformational changes in adsorbed fibrinogen may enhance the exposure of platelet adhesive sites that are inaccessible in solution. To test this hypothesis, mass spectrometric methods were developed to quantify chemical modification of lysine residues following adsorption of fibrinogen to biomaterials. The quantitative method used an internal standard consisting of isotope-labeled fibrinogen secreted by human HepG2 cells in culture. Lysine residues in the internal standard were partially reacted with NHS-biotin. For the experimental samples, normal human fibrinogen was adsorbed to poly(ethylene terephthalate) (PET) particles. The adsorbed fibrinogen was reacted with NHS-biotin and then eluted from the particles. Constant amounts of internal standard were added to sample fibrinogen and analyzed by liquid chromatography/tandem mass spectrometry. Biotinylation of the lysine residue in the platelet-adhesive gamma chain dodecapeptide (GCDP) was quantified by comparison with the internal standard. Approximately 80% of the GCDP peptides were biotinylated when fibrinogen was reacted with NHS-biotin in solution or adsorbed onto PET. These results are generally consistent with previous antibody binding studies and suggest that other regions of fibrinogen may be crucial in promoting platelet adhesion to materials. The results do not directly address but are consistent with the hypothesis that only activated platelets adhere to adsorbed fibrinogen., (Copyright © 2011 Wiley Periodicals, Inc.)

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., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

This research focused on developing a modular poly(ethylene glycol) (PEG) scaffold, assembled from PEG microgels and collagen I, to provide an environment to decouple the chemical and mechanical cues within a three-dimensional scaffold. We first characterized the microgel fabrication process, examining the size, polydispersity, swelling ratio, mesh size and storage modulus of the polymer particles. The resulting microgels had a low polydispersity index, PDI=1.08, and a diameter of ~1.6 μm. The mesh size of the microgels, calculated from the swelling ratio, was 47.53 Å. Modular hydrogels (modugels) were then formed by compacting N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride/N-hydroxysuccinimidyl group-activated microgels with PEG-4arm-amine and 0, 1, 10, or 100 μg ml(-1) collagen. The stiffness (G(∗)) of the modugels was not significantly altered with the addition of collagen, allowing for modification of the chemical environment independent from the mechanical properties of the scaffold. PC12 cell aggregation increased in modugels as collagen concentrations increased and cell viability in modugels was improved over bulk PEG hydrogels. Overall, these results indicate that further exploration of modular scaffolds formed from microgels could allow for a better understanding of the relationship between the chemical and mechanical properties and cellular behavior., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Biomaterial microparticles are commonly utilized as growth factor delivery vehicles to induce chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs). To address whether the presence of microparticles could themselves affect differentiation of MSCs, a 3D co-aggregate system was developed containing an equal volume of human primary bone marrow-derived MSCs and non-degradable RGD-conjugated poly(ethylene glycol) microspheres (PEG-μs). Following TGF-β3 induction, differences in cell phenotype, gene expression and protein localization patterns were found when compared to MSC aggregate cultures devoid of PEG-μs. An outer fibrous layer always found in differentiated MSC aggregate cultures was not formed in the presence of PEG-μs. Type II collagen protein was synthesized by cells in both culture systems, although increased levels of the long (embryonic) procollagen isoforms were found in MSC/PEG-μs aggregates. Ubiquitous deposition of type I and type X collagen proteins was found in MSC/PEG-μs cultures while the expression patterns of these collagens was restricted to specific areas in MSC aggregates. These findings show that MSCs respond differently to TGF-β3 when in a PEG-μs environment due to effects of cell dilution, altered growth factor diffusion and/or cellular interactions with the microspheres. Although not all of the expression patterns pointed toward improved chondrogenic differentiation in the MSC/PEG-μs cultures, the surprisingly large impact of the microparticles themselves should be considered when designing drug delivery/scaffold strategies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Surfaces that resist protein adsorption are important for many bioanalytical applications. Bovine serum albumin (BSA) coatings and multi-arm poly(ethylene glycol) (PEG) coatings display low levels of non-specific protein adsorption and have enabled highly quantitative single-molecule (SM) protein studies. Recently, a method was developed for coating a glass with PEG-BSA nanogels, a promising hybrid of these two low-background coatings. We characterized the nanogel coating to determine its suitability for SM protein experiments. SM adsorption counting revealed that nanogel-coated surfaces exhibit lower protein adsorption than covalently coupled BSA surfaces and monolayers of multi-arm PEG, so this surface displays one of the lowest degrees of protein adsorption yet observed. Additionally, the nanogel coating was resistant to DNA adsorption, underscoring the utility of the coating across a variety of SM experiments. The nanogel coating was found to be compatible with surfactants, whereas the BSA coating was not. Finally, applying the coating to a real-world study, we found that single ligand molecules could be tethered to this surface and detected with high sensitivity and specificity by a digital immunoassay. These results suggest that PEG-BSA nanogel coatings will be highly useful for the SM analysis of proteins.

Recapitulating the elegant structures formed during development is an extreme synthetic and biological challenge. Great progress has been made in developing materials to support transplanted cells, yet the complexity of tissues is far beyond that found in even the most advanced scaffolds. Self-assembly is a motif used in development and a route for the production of complex materials. Self-assembly of peptides, proteins and other molecules at the nanoscale is promising, but in addition, intriguing ideas are emerging for self-assembly of micron-scale structures. In this brief review, very recent advances in the assembly of micron-scale cell aggregates and microgels will be described and discussed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

Macroporous hydrogels may have direct applications in regenerative medicine as scaffolds to support tissue formation. Hydrogel microspheres may be used as drug-delivery vehicles or as building blocks to assemble modular scaffolds. A variety of techniques exist to produce macroporous hydrogels and hydrogel microspheres. A subset of these relies on liquid-liquid two-phase systems. Within this subset, vastly different types of polymerization processes are found. In this review, the history, terminology and classification of liquid-liquid two-phase polymerization and crosslinking are described. Instructive examples of hydrogel microsphere and macroporous scaffold formation by precipitation/dispersion, emulsion and suspension polymerizations are used to illustrate the nature of these processes. The role of the kinetics of phase separation in determining the morphology of scaffolds and microspheres is also delineated. Brief descriptions of miniemulsion, microemulsion polymerization and ionotropic gelation are also included., (Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

A critical element in the formation of scaffolds for tissue engineering is the introduction of concentration gradients of bioactive molecules. We explored the use of poly(ethylene glycol) (PEG) microspheres fabricated via a thermally induced phase separation to facilitate the creation of gradients in scaffolds. PEG microspheres were produced with different densities (buoyancies) and centrifuged to develop microsphere gradients. We previously found that the time to gelation following phase separation controlled the size of microspheres in the de-swollen state, while crosslink density affected swelling following buffer exchange into PBS. The principle factors used here to control microsphere densities were the temperature at which the PEG solutions were reacted following phase separation in aqueous sodium sulfate solutions and the length of the incubation period above the 'cloud point'. Using different temperatures and incubation times, microspheres were formed that self-assembled into gradients upon centrifugation. The gradients were produced with sharp interfaces or gradual transitions, with up to 5 tiers of different microsphere types. For proof-of-concept, concentration gradients of covalently immobilized proteins were also assembled. PEG microspheres containing heparin were also fabricated. PEG-heparin microspheres were incubated with fluorescently labeled protamine and used to form gradient scaffolds. The ability to form gradients in microspheres may prove to be useful to achieve better control over the kinetics of protein release from scaffolds or to generate gradients of immobilized growth factors., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The ability to predict endothelial cell migration rates may aid in the design of biomaterials that endothelialize following implantation. However, the complexity of the signaling response to migration-promoting stimuli such as sphingosine 1-phosphate (S1P) makes such predictions quite challenging. A number of signaling pathways impact S1P-mediated cell migration, including the Akt and Src pathways, which both affect activation of the small GTPase Rac. Rac activation promotes the formation of lamellipodia, and thus should be intimately linked to cell migration rates. In immortalized endothelial cells, expression of proteins that inhibit Akt, Src, and Rac (PTEN, CSK, and beta2-chimaerin, respectively) was decreased using RNA interference, resulting in increases in the basal level of activation of Akt, Src, and Rac. Cells were scrape-wounded and stimulated with 1 microM S1P. The timecourse of Akt, Src, and Rac activation was followed over 2 h in the perturbed cells, while migration into the scrape wound was measured over 6 h. Rac activation at 120 min post-stimulation was highly correlated with the mean migration rate of cells, but only in cells stimulated with S1P. Using partial least squares regression, the migration rate of cells into the scrape wound was found to be highly correlated with the magnitude of the early Akt peak (e.g., 5-15 min post-stimulation). These results demonstrated that biochemical measurements might be useful in predicting rates of endothelial cell migration.

Modular, bioactive, macroporous scaffolds were formed by crosslinking poly(ethylene glycol) (PEG) microspheres around living cells. Hydrogel microspheres were produced from reactive PEG derivatives in aqueous sodium sulfate solutions without the use of surfactants or copolymers. Microspheres were formed following thermally induced phase separation if the gel point was reached prior to extensive coarsening of the PEG-rich domains. Three types of PEG microspheres with different functionalities were used to form scaffolds: one type provided mechanical support, the second type provided controlled delivery of the angiogenesis-promoting molecule, sphingosine 1-phosphate (S1P) and the third type served as a slowly dissolving non-cytotoxic porogen. Scaffolds were formed by centrifuging microspheres in the presence of HepG2 hepatoma cells, resulting in a homogenous distribution of cells. During overnight incubation at 37 degrees C, the microspheres reacted with serum proteins in cell culture medium to stabilize the scaffolds. Within 2 days in culture, macropores formed due to the dissolution of the porogenic PEG microspheres, without affecting cell viability. Gradients in porosity were produced by varying the buoyancy of the porogenic microspheres. Conjugated RGD cell adhesion peptides and the delivery of S1P promoted endothelial cell infiltration through macropores in the scaffolds. The scaffolds presented here differ from previous hydrogel scaffolds in that: (i) cells are not encapsulated in hydrogel; (ii) macropores form in the presence of cells; and (iii) scaffold properties are controlled by the modular assembly of different microspheres that perform distinct functions.

The LCST behavior of poly(ethylene glycol) (PEG) in aqueous sodium sulfate solutions was exploited to fabricate microspheres without the use of other monomers, polymers, surfactants or organic solvents. Reactive PEG derivatives underwent thermally induced phase separation to produce spherical PEG-rich domains that coarsened in size pending gelation, resulting in stable hydrogel microspheres between approximately 1 and 100 microns in size. The time required to reach the gel point during the coarsening process and the extent of crosslinking after gelation both affected the final microsphere size and swelling ratio. The gel point could be varied by pre-reaction of the PEG derivatives below the cloud point, or by controlling pH and temperature above the cloud point. Pre-reaction brought the PEG derivatives closer to the gel point prior to phase separation, while the pH and temperature influenced the rate of reaction. Dynamic light scattering indicated a percolation-to-cluster transition about 3-5 min following phase separation. The mean radius of PEG-rich droplets subsequently increased with time to the 1/4th power until gelation. PEG microspheres produced by these methods with controlled sizes and densities may be useful for the production of modular scaffolds for tissue engineering.

Sphingosine 1-phosphate (S1P) promotes endothelial cell migration in vitro and may potentially impact the endothelialization of implanted biomaterials. However, the effects of S1P on endothelial cells (EC) in flowing blood could be negligible due to preactivation of signaling cascades. We previously developed biomaterials that release S1P and wished to determine through in vitro experiments the extent to which EC respond to S1P added to human platelet poor plasma. We found that addition of 200 nM S1P to platelet poor plasma significantly increased cell migration in two migration models. A lower concentration of S1P added to plasma (100 nM) did not increase endothelial cell migration rates, while the cell migration response was saturated above 200 nM S1P. Expression of the main S1P receptor in EC, S1P(1), was elevated in plasma compared to low serum medium, but addition of VEGF or fluid flow elicited a further increase in S1P(1) mRNA, consistent with the synergistic effects observed between S1P, VEGF, and fluid flow. Thus, sustained delivery of S1P from biomaterials might only enhance endothelial cell migration if the concentration of S1P at the surface of the material stimulated adjacent EC to the same extent as approximately 200 nM S1P added to plasma., (2008 Wiley Periodicals, Inc.)

Late-term thrombosis on drug-eluting stents is an emerging problem that might be addressed using extremely thin, biologically active hydrogel coatings. We report a dip-coating strategy to covalently link poly(ethylene glycol) (PEG) to substrates, producing coatings with approximately <100 nm thickness. Gelation of PEG-octavinylsulfone with amines in either bovine serum albumin (BSA) or PEG-octaamine was monitored by dynamic light scattering (DLS), revealing the presence of microgels before macrogelation. NMR also revealed extremely high end-group conversions prior to macrogelation, consistent with the formation of highly crosslinked microgels and deviation from Flory-Stockmayer theory. Before macrogelation, the reacting solutions were diluted and incubated with nucleophile-functionalized surfaces. Using optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D), we identified a highly hydrated, protein-resistant layer with a thickness of approximately 75 nm. Atomic force microscopy in buffered water revealed the presence of coalesced spheres of various sizes but with diameters less than about 100 nm. Microgel-coated glass or poly(ethylene terephthalate) exhibited reduced protein adsorption and cell adhesion. Cellular interactions with the surface could be controlled by using different proteins to cap unreacted vinylsulfone groups within the coating.

Quantitation of relative or absolute amounts of proteins by mass spectrometry can be prone to large errors. The use of MS/MS ion intensities and stable isotope labeling, which we term stable isotope labeling tandem mass spectrometry (SILT), decreases the effects of contamination from unrelated compounds. We present a software package (SILTmass) that automates protein identification and quantification by the SILT method. SILTmass has the ability to analyze the kinetics of protein turnover, in addition to relative and absolute protein quantitation. Instead of extracting chromatograms to find elution peaks, SILTmass uses only scans in which a peptide is identified and that meet an ion intensity threshold. Using only scans with identified peptides, the accuracy and precision of SILT is shown to be superior to precursor ion intensities, particularly at high or low dilutions of the isotope labeled compounds or with low amounts of protein. Using example scans, we demonstrate likely reasons for the improvements in quantitation by SILT. The appropriate use of variable modifications in peptide identification is described for measurement of protein turnover kinetics. The combination of identification with SILT facilitates quantitation without peak detection and helps to ensure the appropriate use of variable modifications for kinetics experiments.

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.

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.

Aims: Restenosis has been the principal limitation of bare metal stents. Based upon the presumption that platelet and inflammatory cell recruitment initiate neointimal proliferation, we explored a novel polymer coating that reduces cell-stent interactions. The purpose of the present study was to investigate the effect of poly(L-lysine)-graft-poly(ethyleneglycol) (PLL-g-PEG) adsorbed to stent surfaces to reduce neointimal hyperplasia in the porcine restenosis model., Methods and Results: Seven animals were instrumented each with 2 stainless steel stents (15 mm length, 2.5-3.5 mm diameter), randomly implanted in 1 major epicardial coronary artery. One stent was dip-coated with PLL-g-PEG, whereas the other stent served as the uncoated control stent. All animals were sacrificed after 6 weeks for histological examination. Neointimal hyperplasia was significantly less (-51%) in the PLL-g-PEG-coated stents (1.15 +/- 0.59 mm2) than in the uncoated control stents (2.33 +/- 1.01 mm2; p < 0.001). Conversely, lumen size was larger in the PLL-g-PEG-coated stents (2.91 +/- 1.17 mm2) than in the uncoated stents (2.04 +/- 0.64 mm2; p < 0.001). High magnification histomorphologic examination revealed no signs of inflammation or thrombus formation in either stent group., Conclusions: Polymeric steric stabilization of stents with PLL-g-PEG significantly reduces neointimal hyperplasia in the porcine restenosis model. Reduction of cell-stent interactions mediated by PLL-g-PEG appear to improve biocompatibility of stainless steel stents without evidence of adverse inflammatory or prothrombotic effects.

While protein growth factors promote therapeutic angiogenesis, delivery of lipid factors such as sphingosine 1-phosphate (S1P) may provide better stabilization of newly formed vessels. We developed a biomaterial for the controlled delivery of S1P, a bioactive lipid released from activated platelets. Multiarm poly(ethylene glycol)-vinyl sulfone was cross-linked with albumin, a lipid-transporting protein, to form hydrogels. The rate of S1P release from the materials followed Fickian kinetics and was dependent upon the presence of lipid carriers in the release solution. Delivery of S1P from RGD-modified hydrogels increased the cell migration speed of endothelial cells growing on the materials. The materials also induced angiogenesis in the chorioallantoic membrane assay. Our data demonstrate that the storage and release of lipid factors provides a new route for the induction of angiogenesis by artificial materials.

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., ((c) 2005 Wiley Periodicals, Inc.)

The rational design of drug delivery systems requires the ability to predict the environment-specific responses of target cells to the delivered drug. Here we describe the in vitro effects of fluid shear stress, vascular endothelial growth factor (VEGF), and sphingosine 1-phosphate (S1P) on the migration of human umbilical vein endothelial cells (HUVEC). Endothelial cell migration into a scrape wound was enhanced in S1P- or VEGF-stimulated HUVEC by the addition of fluid shear stress. In both cases, scrape wound closure rates were near a maximal value that was not exceeded when cells were exposed to all three factors. We also found that cell migration into a scrape wound due to S1P stimulation was correlated with the S1P1 mRNA concentration, in systems where cell migration was not already near maximal. The present work represents our initial steps toward predicting cell migration based upon the activation state of the receptors and enzymes involved in the chemokinetic response. These results also illustrate the importance of context-dependent analysis of cell signaling cascades.

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.

The purpose of this short communication is to describe the reaction rate for the removal of the N-methyltrityl (Mtt) protecting group that is used in solid-phase peptide synthesis for the production of branched and cyclic peptides. The reaction rate was observed to follow zero-order kinetics, and we suggest the optimal conditions for the removal of the Mtt group in batchwise synthesis.

A new chemical cross-linking scheme was utilized for the formation of degradable poly(ethylene glycol) hydrogels suitable for the delivery of protein drugs. An aqueous solution containing a PEG-multiacrylate and solid particles of albumin was mixed with an aqueous solution containing a PEG-dithiol, rapidly producing a cross-linked hydrogel through a Michael-type addition reaction. For some formulations, it was observed that about 65% of the incorporated protein was released with zero-order kinetics over a period of about 4 days. By changing the functionality of the cross-linker, the release of protein could even be delayed for about 4 days, followed by zero-order release. The mechanism for release appeared to be a combination of slow dissolution of protein in the presence of PEG and hindered diffusion of protein through the gel. The cross-linking of the gels was studied rheometrically, and the hydrolytic degradation of the gels was characterized by measuring the swelling of the gels. Biochemical analysis of the released proteins demonstrated that the polymers reacted with each other, but not with proteins. Utilizing the Flory-Rehner and Peppas-Merrill equations, a framework for modeling the protein release from the gels is described.

PEG-diacrylamide was synthesized and utilized to make materials for tissue engineering. Acrylamide groups readily react with thiol groups, and peptides containing a single thiol group were coupled to the PEG-diacrylamide in aqueous solution at room temperature in about 2 h. If only a portion of the acrylamide groups were targeted for reaction with peptide, sufficient amounts of PEG-diacrylamide remained to be polymerized by free-radical mechanisms via photoinitiation. The photopolymerization step can be performed in contact with cells, providing a means to produce bioactive scaffolds for tissue engineering. The photopolymerization conditions and precursor composition greatly affect the stiffness of the materials, which subsequently affected cell spreading. The kinetics and extent of cell spreading on the bioactive materials were measured and compared to cell spreading on tissue culture polystyrene. Although the PEG materials resist protein adsorption, the experiments suggest that the cells can secrete extracellular matrix that can adhere to the gels.

Postsurgical adhesions represent a common complication following a variety of surgical procedures. We sought to develop and evaluate a water-soluble polymer that could self-assemble onto tissue surfaces, forming a barrier on the surface. A copolymer was synthesized so as to contain two components: one component adsorbed to the tissue surface, and the other created a steric barrier, thereby preventing cell interactions with the tissue surface, and perhaps altering the wound-healing response that leads to the formation of fibrous adhesions. The component selected for tissue binding was a water-soluble polycation, poly-L-lysine, which can bind to negative sites on glycoproteins, proteoglycans, and cells; and the component selected for steric stabilization was polyethylene glycol, a nonionic polymer that interacts poorly with proteins. Efficacy of lavage with an aqueous solution of the copolymer for the prevention of postsurgical abdominopelvic adhesions was assessed following a standard electrocautery injury of the uterine horns of rats. The copolymer resulted in an 88% reduction in the extent of adhesions that formed. In vitro studies designed to investigate the mechanism of this efficacy indicated that the copolymer may both hinder cell-tissue adhesive interactions and alter the process of fibrin formation.

Background: We present the synthesis, characterization and initial structure-function analysis of a new class of bioactive agent that allows the application of techniques from colloid science to biological surfaces. Stable colloidal suspensions can be generated by immobilizing a dense brush of soluble polymer at the colloidal surface, creating a zone protected against the adhesion of approaching particles, a phenomenon termed polymeric steric stabilization. This is often accomplished for aqueous colloidal dispersions using adsorbing block copolymers. We demonstrate that water-soluble block copolymers can be designed to adsorb onto heterogeneous biological surfaces and block cell-cell and cell-surface adhesion, using polymer compositions and architectures that are quite different from surfactants used for stabilizing nonbiological colloidal dispersions., Results: Comb copolymers were synthesized having polycationic backbones (poly-L-lysine, PLL), serving as the anchor for binding to the net negatively charged biological surfaces, grafted with water-soluble polynonionic chains (polyethylene glycol, PEG), to block biological recognition, producing PLL-graft-PEG copolymers. Specific copolymers were found to sterically stabilize red blood cells from lectin-induced hemagglutination and fibroblasts from adhesion to fibronectin-coated surfaces. The polymer design principles, which appear to be unique for adsorption to heterogeneous biological surfaces, require the use of very high molecular weight comb copolymers, perhaps because anionic sites are non-uniformly distributed on biological surfaces, and the ability of larger copolymers to span between highly anionic sites., Conclusions: Water-soluble copolymers were produced that can block recognition at biological surfaces, on the basis of nonspecific physicochemical phenomena rather than specific biochemical interactions.

Novel artificial extracellular matrices were synthesized in the form of semi-interpenetrating polymer networks containing copolymers of poly(ethylene glycol) and acrylic acid (PEG-co-AA) grafted with synthetic bioadhesive peptides onto exposed carboxylic acid moieties. These substrates were very resistant to cell adhesion, but when they were grafted with adhesive peptides they were highly biospecific in their ability to support cell adhesion. Extensive preadsorption of adhesive proteins or peptides did not render these materials cell adhesive; yet covalent grafting of adhesive peptides did render these materials highly cell adhesive even in the absence of serum proteins. Polymer networks containing immobilized PEG-co-AA were grafted with peptides at densities of 475 +/- 40 pmol/cm(2). Polymer networks containing immobilized PEG-co-AA N-terminally grafted with GRGDS supported cell adhesion efficiencies of 42 +/- 4% 4 h after seeding and became confluent after 12 h. These cells displayed cell spreading and cytoskeletal grafted with inactive control peptides (GRDGS, GRGES, or no peptide) supported cell adhesion efficiencies of 0 +/- 0%, even when challenged with high seeding densities (to 100,000 cell/cm(2)) over 14 days. These polymer networks are suitable substrates to investigate in vitro cell-surface interactions in the presence of serum proteins without nonspecific protein adsorption adhesion signals other than those immobilized for study.