A multicenter, randomized, double-blind, placebo-controlled ascending dose study to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamic (PD) effects of Posiphen in subjects with early Alzheimer's Disease.

BACKGROUND: Amyloid beta protein (Aß) is a treatment target in Alzheimer's Disease (AD). Lowering production of its parent protein, APP, has benefits in preclinical models. Posiphen, an orally administered small molecule, binds to an iron-responsive element in APP mRNA and decreases translation of APP and Aß. To augment human data for Posiphen, we evaluated safety, tolerability and pharmacokinetic and pharmacodynamic (PD) effects on Aß metabolism using Stable Isotope Labeling Kinetic (SILK) analysis. METHODS: Double-blind phase 1b randomized ascending dose clinical trial, at five sites, under an IRB-approved protocol. Participants with mild cognitive impairment or mild AD (Early AD) confirmed by low CSF Aß42/40 were randomized (within each dose arm) to Posiphen or placebo. Pretreatment assessment included lumbar puncture for CSF. Participants took Posiphen or placebo for 21-23 days, then underwent CSF catheter placement, intravenous infusion of 13C6-leucine, and CSF sampling for 36 h. Safety and tolerability were assessed through participant reports, EKG and laboratory tests. CSF SILK analysis measured Aß40, 38 and 42 with immunoprecipitation-mass spectrometry. Baseline and day 21 CSF APP, Aß and other biomarkers were measured with immunoassays. The Mini-Mental State Exam and ADAS-cog12 were given at baseline and day 21. RESULTS: From June 2017 to December 2021, 19 participants were enrolled, randomized within dose cohorts (5 active: 3 placebo) of 60 mg once/day and 60 mg twice/day; 1 participant was enrolled and completed 60 mg three times/day. 10 active drug and 5 placebo participants completed all study procedures. Posiphen was safe and well-tolerated. 8 participants had headaches related to CSF catheterization; 5 needed blood patches. Prespecified SILK analyses of Fractional Synthesis Rate (FSR) for CSF Aß40 showed no significant overall or dose-dependent effects of Posiphen vs. placebo. Comprehensive multiparameter modeling of APP kinetics supported dose-dependent lowering of APP production by Posiphen. Cognitive measures and CSF biomarkers did not change significantly from baseline to 21 days in Posiphen vs. placebo groups. CONCLUSIONS: Posiphen was safe and well-tolerated in Early AD. A multicenter SILK study was feasible. Findings are limited by small sample size but provide additional supportive safety and PK data. Comprehensive modeling of biomarker dynamics using SILK data may reveal subtle drug effects. TRIAL REGISTRATION: NCT02925650 on clinicaltrials.gov (registered on 10-24-2016).

A multicenter, randomized, double-blind, placebo-controlled ascending dose study to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamic (PD) effects of Posiphen in subjects with Early Alzheimer's Disease.

Background: Amyloid beta protein (Aß) is a treatment target in Alzheimer's Disease (AD). Lowering production of its parent protein, APP, has benefits in preclinical models. Posiphen binds to an iron-responsive element in APP mRNA and decreases translation of APP and Aß. To augment human data for Posiphen, we evaluated safety, tolerability and pharmacokinetic and pharmacodynamic (PD) effects on Aß metabolism using Stable Isotope Labeling Kinetic (SILK) analysis. Methods: Double-blind phase 1b randomized ascending dose clinical trial, at five sites, under an IRB-approved protocol. Participants with mild cognitive impairment or mild AD (Early AD) with positive CSF biomarkers were randomized (within each dose arm) to Posiphen or placebo. Pretreatment assessment included lumbar puncture for CSF. Participants took Posiphen or placebo for 21-23 days, then underwent CSF catheter placement, intravenous infusion of 13C6-leucine, and CSF sampling for 36 hours. Safety and tolerability were assessed through participant reports, EKG and laboratory tests. CSF SILK analysis measured Aß40, 38 and 42 with immunoprecipitation-mass spectrometry. Baseline and day 21 CSF APP, Aß and other biomarkers were measured with immunoassays. The Mini-Mental State Exam and ADAS-cog12 were given at baseline and day 21. Results: From June 2017 to December 2021, 19 participants were enrolled, in dose cohorts (6 active: 2 placebo) of 60 mg once/day and 60 mg twice/day; 1 participant was enrolled and completed 60 mg three times/day. 10 active drug and 5 placebo participants completed all study procedures. Posiphen was safe and well-tolerated. 8 participants had headaches related to CSF catheterization; 5 needed blood patches. Prespecified SILK analyses of Fractional Synthesis Rate (FSR) for CSF Aß40 showed no significant overall or dose-dependent effects of Posiphen vs. placebo. Comprehensive multiparameter modeling of APP kinetics supported dose-dependent lowering of APP production by Posiphen. Cognitive measures and CSF biomarkers did not change significantly from baseline to 21 days in Posiphen vs placebo groups. Conclusions: Posiphen was safe and well-tolerated in Early AD. A multicenter SILK study was feasible. Findings are limited by small sample size but provide additional supportive safety and PK data. Comprehensive modeling of biomarker dynamics using SILK data may reveal subtle drug effects. Trial registration: NCT02925650 on clinicaltrials.gov.

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.

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.

Amyloid-ß Plaques in Clinical Alzheimer's Disease Brain Incorporate Stable Isotope Tracer In Vivo and Exhibit Nanoscale Heterogeneity.

Alzheimer's disease (AD) is a neurodegenerative disorder with clinical manifestations of progressive memory decline and loss of executive function and language. AD affects an estimated 5.3 million Americans alone and is the most common form of age-related dementia with a rapidly growing prevalence among the aging population-those 65 years of age or older. AD is characterized by accumulation of aggregated amyloid-beta (Aß) in the brain, which leads to one of the pathological hallmarks of AD-Aß plaques. As a result, Aß plaques have been extensively studied after being first described over a century ago. Advances in brain imaging and quantitative measures of Aß in biological fluids have yielded insight into the time course of plaque development decades before and after AD symptom onset. However, despite the fundamental role of Aß plaques in AD, in vivo measures of individual plaque growth, growth distribution, and dynamics are still lacking. To address this question, we combined stable isotope labeling kinetics (SILK) and nanoscale secondary ion mass spectrometry (NanoSIMS) imaging in an approach termed SILK-SIMS to resolve plaque dynamics in three human AD brains. In human AD brain, plaques exhibit incorporation of a stable isotope tracer. Tracer enrichment was highly variable between plaques and the spatial distribution asymmetric with both quiescent and active nanometer sub-regions of tracer incorporation. These data reveal that Aß plaques are dynamic structures with deposition rates over days indicating a highly active process. Here, we report the first, direct quantitative measures of in vivo deposition into plaques in human AD brain. Our SILK-SIMS studies will provide invaluable information on plaque dynamics in the normal and diseased brain and offer many new avenues for investigation into pathological mechanisms of the disease, with implications for therapeutic development.

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.

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.

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.

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.

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.

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.

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.

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.

Exposure of the lysine in the gamma chain dodecapeptide of human fibrinogen is not enhanced by adsorption to poly(ethylene terephthalate) as measured by biotinylation and mass spectrometry.

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.

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.

Changes of chondrocyte expression profiles in human MSC aggregates in the presence of PEG microspheres and TGF-ß3.

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-ß3 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-ß3 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.