Coulter Foundation Research Articles

Patient-Reported Quality of Life (QoL) Following CTL019 Infusion in Adult Patients (pts) with Relapsed/Refractory (r/r) Diffuse Large B-Cell Lymphoma (DLBCL)

Patient-Reported Quality of Life (QoL) Following CTL019 Infusion in Adult Patients (pts) with Relapsed/Refractory (r/r) Diffuse Large B-Cell Lymphoma (DLBCL)

β-N-methylamino-l-alanine analysis in the brains of patients with Kii ALS/PDC.

The Kii Peninsula of Japan and the island of Guam are known as high-incidence areas of amyotrophic lateral sclerosis and parkinsonism–dementia complex (ALS/PDC). The disorder is clinically characterized by a variable presentation of parkinsonism, dementia, and motor neuron symptoms and pathologically by tau protein deposits in the CNS.1 Both genetic and environmental factors are implicated in ALS/PDC pathogenesis. Exposure to β-N-methylamino-l-alanine (BMAA), a neurotoxin produced by cyanobacteria, has been proposed as a risk factor for ALS/PDC in Guam.2 It is not known whether Kii ALS/PDC shares the same etiology as Guam ALS/PDC. To address these issues, we conducted BMAA analyses of brain samples from 5 patients with Kii ALS/PDC using high-performance liquid chromatography (HPLC) and liquid chromatography–tandem mass spectrometry (LC/MS/MS). Acknowledgment: The authors thank Junko Karita for clerical assistance and Enago ( [ enago.jp ][1] ) for the English language review. The authors also thank the H.L. Castle Foundation, the W. F. Coulter Foundation, and the Margaret Q. Landenberger Foundation, as well as C. Childs, K. Farkas, and L. Atherton for research funding. [1]: http://www.enago.jp/

Impact of post‐transplant lymphoproliferative disorder subtype on survival

Abstract Introduction Post-transplant lymphoproliferative disease (PTLD) arises in the setting of immunosuppression following solid organ transplantation (SOT) or hematopoietic stem cell transplantation. PTLD incidence ranges from 1 to 20% in SOT; mortality estimates of >30% make this entity a significant life-threatening complication of transplantation. PTLD is broadly categorized into four groups: early; polymorphic; monomorphic, including T/natural killer cell neoplasms and most B cell neoplasms; and classical Hodgkin lymphoma. The heterogeneity of PTLD histology, compounded by relative disease rarity and absence of a standard treatment approach, has made evaluating clinical outcomes in specific patient populations difficult. We analyzed data from a systematic review of the literature to investigate the impact of PTLD histologic subtype on survival in a large dataset. Methods Case series were identified on PubMed using the search terms “post-transplant lymphoproliferative disorder/disease,” “PTLD”, and “solid organ transplantation,” with additional publications identified through reference lists of these papers. Inclusion criteria were: 1) articles published in English between January 1, 1974 and July 1, 2016 with pathology diagnosis and survival details; 2) adult cases following SOT. Patient characteristics, immunosuppressive regimen, treatment, survival, and follow-up time of 306 cases were extracted from 94 articles and combined with 11 cases from our institution. Patients with recorded subtype information were included in survival analysis. Kaplan-Meier analysis compared overall survival (OS) for four major subtypes (polymorphic, monomorphic B cell, monomorphic T cell, and Hodgkin-type neoplasms), and for four subgroups of B cell neoplasms. Univariate and multivariable Cox proportional hazard regression analyses were performed to identify predictors of OS for each subtype and B cell subgroup. Results Of 234 cases with available subtype information, 52.1% were monomorphic B cell neoplasms, of which DLBCL was the most frequent subtype (n=59, 48.3% of B neoplasms); 28.6% were monomorphic T cell neoplasms, 10.6% were Hodgkin-type, and 4.3% were polymorphic. OS was significantly different between monomorphic T cell neoplasms (median 9 months) and polymorphic, monomorphic B cell, and Hodgkin-type neoplasms, for which median OS was not reached (p = 0.0001, Figure 1a). Significant differences in OS among B-subgroups were not detected, but there was a trend towards decreased survival in Burkitt-type PTLD (Figure 1b). Kidney transplant and treatment with reduction of immunosuppression were associated with increased OS in multivariable analysis in B cell neoplasms. Immunosuppression with azathioprine was associated with decreased OS in T cell neoplasms, while treatment with radiotherapy was associated with improved OS in that subtype. Conclusions Histologic subtype represents an important factor in PTLD prognosis, with T cell monomorphic subtype exhibiting a particularly poor OS. The possibility of lower survival in certain subsets of B cell PTLD should be explored in future studies and suggests the need for subtype-specific treatment strategies to improve outcomes. Download : Download high-res image (92KB) Download : Download full-size image Disclosures Flowers: Onyx: Research Funding; TG Therapeutics: Research Funding; Research to Practice: Research Funding; Genentech/Roche: Consultancy, Research Funding; Prime Oncology: Research Funding; Burroughs Welcome Fund: Research Funding; OptumRx: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Eastern Cooperative Oncology Group: Research Funding; Janssen Pharmaceutical: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Celgene: Consultancy, Research Funding; Millennium/Takeda: Research Funding; National Institutes Of Health: Research Funding; National Cancer Institute: Research Funding; Acerta: Research Funding; Clinical Care Options: Research Funding; Infinity: Research Funding; Spectrum: Consultancy; V Foundation: Research Funding; Abbvie: Consultancy, Research Funding; Educational Concepts: Research Funding; Gilead: Consultancy. Waller: PRA: Consultancy; National Institutes of Health: Research Funding; AMGEN: Consultancy; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Katz Foundation: Research Funding; Chimerix: Equity Ownership; Celldex: Consultancy; Coulter Foundation: Research Funding; Helocyte: Consultancy; Cerus: Equity Ownership; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding.

WE‐DE‐201‐07: Measurement of Real‐Time Dose for Tandem and Ovoid Brachytherapy Procedures Using a High Precision Optical Fiber Radiation Detector

Purpose:Development of a novel on‐line dosimetry tool is needed to move toward patient‐specific quality assurance measurements for Ir‐192 HDR brachytherapy to verify accurate dose delivery to the intended location. This work describes the development and use of a nano‐crystalline yttrium oxide inorganic scintillator based optical‐fiber detector capable of acquiring real‐time high‐precision dose measurements during tandem and ovoid (T&O) gynecological (GYN) applicator Ir‐192 HDR brachytherapy procedures.Methods:An optical‐fiber detector was calibrated by acquiring light output measurements in liquid water at 3, 5, 7, and 9cm radial source‐detector‐distances from an Ir‐192 HDR source. A regression model was fit to the data to describe the relative light output per unit dose (TG‐43 derived) as a function of source‐detector‐distance. Next, the optical‐fiber detector was attached to a vaginal balloon fixed to a Varian Fletcher‐Suit‐Delclos‐style applicator (to mimic clinical setup), and localized by acquiring high‐resolution computed tomography (CT) images. To compare the physical point dose to the TPS calculated values (TG‐43 and Acuros‐BV), a phantom measurement was performed, by submerging the T&O applicator in a liquid water bath and delivering a treatment template representative of a clinical T&O procedure. The fiber detector collected scintillation signal as a function of time, and the calibration data was applied to calculate both real‐time dose rate, and cumulative dose.Results:Fiber cumulative dose values were 100.0cGy, 94.3cGy, and 348.9cGy from the tandem, left ovoid, and right ovoid dwells, respectively (total of 443.2cGy). A plot of real time dose rate during the treatment was also acquired. The TPS values at the fiber location were 458.4cGy using TG‐43, and 437.6cGy using Acuros‐BV calculated as Dm,m (per TG‐186).Conclusion:The fiber measured dose value agreement was 3% vs TG‐43 and −1% vs Acuros‐BV. This fiber detector opens up new possibilities for performing patient‐specific quality assurance for Ir‐192 HDR GYN procedures.Funding from Coulter Foundation, Duke Bio‐medical Engineering. Company is being created around the detector technology. Duke holds patents on the technology.

Electrospun poly(dimethyl siloxane)-based meshes as a platform for t cell expansion

Event Abstract Back to Event Electrospun poly(dimethyl siloxane)-based meshes as a platform for t cell expansion Alex Dang1, Sarah E. De Leo1, Danielle R. Bogdanowicz1, Helen H. Lu1 and Lance C. Kam1 1 Columbia University, Biomedical Engineering, United States Introduction: Recent advances in the genetic manipulation of T cells have led to striking successes in immunotherapy of cancer. However, advances in cell production and manufacturing are needed to fully realize the power of this approach. The current gold standard for initiating clinical ex vivo expansion of T cell populations consists of rigid (GPa) polystyrene beads presenting activating antibodies to CD3 and CD28. Recent work by our lab demonstrated that improved human T cell stimulation can be achieved using softer poly(dimethyl siloxane) (PDMS) substrates (~30 kPa – 2 MPa)[1]m[2]. In this report, we leverage this mechanosensing property of T cells in a fiber-based mesh system to improve human T cell expansion on a clinically relevant scale. This format preserves the high surface area provided by beads while reducing the risks associated with incomplete removal of these structures from the final cell product. We herein examine the effect of composition, rigidity, fiber density, and pore size of electrospun polymer fibers on long-term (19 days) T cell expansion on clinically relevant scales. Materials and Methods: Fibrous meshes were synthesized via electrospinning of NuSil MED-4086 PDMS mixed with poly(ε-caprolactone) (PCL) in a 3:1 v/v dichloromethane (DCM) / N,N-dimethylformamide (DMF) solution at 10 – 12 kV onto a grounded collection electrode with a working distance between 10-12 cm. Polymer concentration was varied from 19 to 50% wt/wt. Meshes were coated with goat-anti-mouse IgG linkers followed by a 1:4 ratio of mouse IgG-anti-human CD3 (OKT3) to mouse IgG-anti-human CD28 (9.3) (2 h, 23 °C). Cells were subjected to a carboxy-fluorescein succinimidyl ester (CFSE) assay and seeded at 1 million cells / mL at either small-scale (1 mL) or clinical scale (15-20 mL) volumes. Cell proliferation studies were performed in vitro with human naïve CD4+/CD8+ T cells as previously described[1]. Assays for CD107b expression and IFNγ secretion were performed upon completion of blast phase and in vitro re-stimulation for 4 and 12 hours, respectively. Assays for memory markers CD45RO and CCR7 were performed following re-stimulation. Results and Discussion: Scanning electron microscopy (SEM) revealed tunable pore sizes and fiber densities as a function of polymer composition. Long-term culture for 17-19 days coupled with approximately 9.8 maximum doublings for PDMS/PCL meshes indicated a robust system for expansion of T cells. Expansions on experimental meshes yielded up to 3.2 and 4 times more cells compared to Dynabead® and pure PCL mesh controls, respectively (see fig. 1). Flow cytometry revealed higher levels of proliferation on experimental meshes than on the current gold standard and pure PCL controls. Assays for CD107b / CD45RO / CCR7 expression and IFNγ secretion showed PDMS/PCL mesh-expanded cell activation levels on par with or higher than cells expanded on the current gold standard (see fig. 2). Memory marker analysis revealed high levels of central memory T cells after re-stimulation (data not shown). Conclusion: We have shown that electrospun, antibody-functionalized PDMS/PCL fibrous meshes with microscale diameters and pore sizes can increase T cell proliferation. Cells produced yielded substantially higher population doublings and were shown to have equal or better levels of effector cell function than the current gold standard. This platform holds significant promise in the ex vivo expansion of T cells. National Institutes of Health (R01AI088377); Wallace H. Coulter Foundation; National Science Foundation GRFP; Columbia University Medical Center Flow Cytometry Core

The effect of biomimetic proteoglycans on collagen self-assembly

Event Abstract Back to Event The effect of biomimetic proteoglycans on collagen self-assembly Tao Yang1, Kate Wofford2, Parmpuneet Kaur2, Carli Moorehead1, Joseph Sincavage2, Katsiaryna Prudnikova1, Adrian C. Shieh2 and Michele Marcolongo1 1 Drexel University, Department of Material Science and Engineer, United States 2 Drexel University, School of Biomedical Engineering, Science & Health Systems, United States Introduction: Collagen is one of the leading components in extracellular matrix (ECM), providing durability, structural integrity, and functionality for many tissues. Excessive collagen degradation has been associated with a number of disorders, such as arthritis, atherosclerotic heart disease, and tumor metastasis[1]. Materials capable of preserving tissue function and preventing tissue degradation have attracted attentions intensively. Recently, we developed a novel, biocompatible, semi-synthetic proteoglycan Biomimetic Aggrecan (BA) with a high (BA250) and low (BA10) molecular weight. BA250 and BA10 consist of an enzymatically resistant synthetic polymer core (poly(acrylic acid)) and natural chondroitin sulfate bristles arranged in a three-dimensional bottle-brush configuration similar to the natural proteoglycan aggrecan[2]. We examined the impact of BA on collagen gelation kinetics and quantified the fibril characteristics during collagen self-assembly. Materials and Methods: Soluble type 1 rat-tail was neutralized with 1N NaOH and mixed with appropriate amounts of BA and 1xPBS for a final concentration of 2.0 mg/mL. Collagen gels were kept at 4°C for 30 minutes, followed up by incubation at 37°C for 2 hours. All gels were imaged on an Olympus FV1000-laser scanning confocal with a 60x oil-emersion objective at 488nm. Fibrils were traced in each image manually using ImageJ. After tracing, the data of fibril length was binned in 10μm increments to determine length distributions in each gel type. Average number of fibrils was obtained by summing fibrils tracings within each treatment. For fibrillogenesis study, after neutralization for 30 minutes, collagen solutions were transferred into a 96 well plate and incubated in the Tecan spectrometer (Infinite M200) at 37°C for 1 hour. The absorbance was measured at a wavelength of 313 nm every minute during incubation (n=5). Results and Discussion: Confocal images (Fig.1A) showed significant variance with the addition of BA. Turbidity measurement during gel incubation revealed a prolonged period of gelation with addition of BA10 or BA250 indicating that addition of BA can affect fibriollegensis (Fig.1B).Compared to collagen-only gels, BA10-gels slightly differed in length distribution while BA250-gels showed a trend towards forming longer fibrils as BA250 concentration increased (Fig.1C). Average number of fibrils was larger in BA-gels with lower concentrations (0.01mg/ml and 0.05mg/mL) than those with the highest concentration (0.1mg/mL), indicating lower concentrations of BA create more fibrils while higher concentrations of BA create longer fibrils. Conclusion: A three-dimensional collagen scaffold was fabricated and collagen fibril organization was modulated by two aggrecan mimics developed in our lab, indicating that collagen self-assembly could be structurally altered by incorporation of a biomimetic proteoglycan, such as BA. Therefore, biomimetic proteoglycan is a promising material that could be used to tune collagen scaffold characteristics for tissue engineering applications. Coulter Foundation

Cellulose based micro- nano structured scaffolds for bone regeneration

Event Abstract Back to Event Cellulose based micro- nano structured scaffolds for bone regeneration Aja Aravamudhan1, 2 and Sangamesh G. Kumbar1, 2, 3, 4 1 University of Connecticut Health Center, Institute for Regenerative Engineering, United States 2 University of Connecticut Health Center, Department of Orthopeadic Surgery, United States 3 University of Connecticut Health Center, Department of Materials Science and Engineering, United States 4 University of Connecticut Health Center, Department of Biomedical Engineering, United States Introduction: Several polymer based scaffold systems have been used as an alternative to traditional autografts and allografts to repair non-healing defects of the bone. These materials fail to provide required osteoconductive, osteoinductive, and osteointegrative properties in line with autografts. Natural polymers have the advantage of being similar to biological macromolecules and elicit favorable tissue healing responses, in contrast to synthetic polymers that are used conventionally in scaffolds for bone regeneration. In this study, we present the characterization of cellulose acetate based scaffolds in comparison to synthetic poly(lactic-co-glycolic acid) (PLGA) scaffolds with respect to their ability to promote hMSC’s progression into osteoblastic lineage in vitro, biocompatibility in a rat subcutaneous implantation model and bone healing capability in a mouse calvarial defect model. Materials and Methods: Cellulose acetate (CA) (Sigma-Aldrich, 30kD) and (PLGA) (P) microparticles were prepared using emulsion solvent evaporation method. Solvent sintering technique[1],[2] was employed to prepare CA scaffolds and heat sintering was used for PLGA. Composite scaffolds of CA (CAc) PLAGA (Pc) were prepared by coating 0.1 % collagen solution on the scaffolds. In-vitro studies, using hMSCs in basal media (BM) and osteogenic media (OM), were conducted by seeding 500,000 cells per tablet type scaffold. Osteoblastic differentiation was monitored by alkaline phosphate activity assay and mineralization along with change in osteogenic gene expression (RUNX2, Col1, Col3, and BSP after 21 days). Further, CA, CAc and PLGA scaffolds were implanted subcutaneously in rats to determine the biocompatibility of the materials by contrasting the immune responses between the groups using histological staining. The scaffolds seeded with bone marrow stomal cells from donor mice (Col 3.6 cyan+) were implanted in critical sized (3.5mm dia) calvarial defect in mice (Col 3.6 GFP+) and its ability to heal bone was evaluated by histological staining. Results and Discussion: Osteogenic genes such as RUNX2, collagen1 and BSP showed greater expression on the CA and CAc groups in contrast to the PLGA groups (Figure 1.a.). The CA and CAc scaffolds showed greater progression of osteogenic phenotype as seen by greater protein levels of Col1 and BSP (Figure 1.b.) and mineralization of osteoinduced hMSCs on these scaffolds (Figure 1.c.). Among the scaffolds implanted subcutaneously (Figure 2.a.), the foreign body response (FBR) reduced significantly over time on the CA groups, while the FBR response increased over time on PLGA (Figure 2.b.). These results indicate the long-term biocompatibility of CA groups over synthetic PLGA. Finally, CA scaffolds seeded with MSCs showed uniform bone formation in contrast to PLGA scaffolds that had islands of new bone formation (Figure 2.c.). The greater hydrophilic and biomimetic nature of CA may be attributed to these differences. Conclusions: The CA and CAc scaffolds induced and maintained osteoblastic differentiation of the seeded hMSCs. The biocompatibility and cellularity of CA increased over time, while that of PLGA decreased over time. The stable and more hydrophilic properties of CA could be attributed to these results. Finally the bone formation by CA scaffolds was uniformly well distributed through the scaffold than PLGA, reflecting the superior osteogenic performace of the polysaccharide-based scaffolds. Hence CA based polysaccharide platform may be a viable alternative to synthetic polymers like PLGA in bone tissue engineering. Coulter Foundation, National Science Foundation (IIP-1355327, IIP- 1311907 and EFRI-1332329); Raymond and Beverly Sackler Center

Molecular engineering of the urethra for treatment of stress incontinence using a novel biomimetic aggrecan ex vivo and in vivo

Event Abstract Back to Event Molecular engineering of the urethra for treatment of stress incontinence using a novel biomimetic aggrecan ex vivo and in vivo Alicia Kriete1*, Natasha Ginzburg2, Nima Shah2, Richard Huneke2, Emily Reimold2, Katsiaryna Prudnikova1*, Owen Montgomery2* and Michele Marcolongo1* 1 Drexel University, Materials Science and Engineering, United States 2 Drexel University, College of Medicine, United States Introduction: Stress urinary incontinence (SUI) is the involuntary leakage of urine through urethra during physical activity such as walking, exercising, lifting, and in severe cases, even laughing and coughing, affecting about 56% of postmenopausal women[1]. SUI is associated with the decrease in volume and compliance of urethral tissue[2][3]. Injecting bulking agents into urethral tissue is an underutilized treatment option for SUI, primarily because these treatments only last for up to 1 year due to migration of the bulking agents away from the urethra[4]. Here we report our initial findings of using biomimetic aggrecan (BA) to molecularly engineer urethral tissue ex vivo and in vivo with the goal of increasing urethral compliance by bringing more hydration to the tissue because of the negatively charged BA molecules incorporated into the urethra extracellular matrix (ECM). Materials and Methods: BA was synthesized via a “grafting-to” method in which natural chondroitin sulfate bristles were coupled to functional groups on an enzymatically resistant polyacrylic acid core (250 kDa) in a 3D bottle brush configuration[5]. Porcine urethras (Animal Technologies, Inc.) were purchased and injection and radial expansion techniques were developed to test the effect of BA on tissue compliance ex vivo. 25 gauge needles were utilized to inject porcine cadavers transurethrally with 0.5 ml BA solutions at 3 sites (120 ° apart), approximately 1-2 mm deep into tissue. BA concentrations of 25 and 50 mg/ml in 1XPBS were tested, and compared to non-injected control tissue (n=4). Radial expansion testing (RET) was performed by transurethrally inserting a balloon catheter into injected porcine urethra. In order to study integration of BA into the ECM, fluorescently-tagged BA was injected into porcine urethra and imaged using confocal microscopy. Micro-CT imaging was performed to study volume change of BA-injected porcine tissue. Biocompatibility of BA was tested in vivo by transurethrally injecting New Zealand White rabbits (n=6) with 0.5 ml of BA solution using a cystoscope (BA concentrations of 50 mg/ml and 200 mg/ml). Initial assessment of in vivo residence time was studied with fluorescently- labeled BA inejctions (figure 1). Urethras were harvested after 1 and 6 weeks, and histological analysis was performed. Results and Discussion: RET results show there is a statistically significant reduction in pressure of BA-injected samples; ~20% decrease (p<0.05) and 35% decrease (p<0.01) for 25 and 50 mg/ml BA injections, respectively indicating increased compliance was achieved, potentially allowing for better sealing of the urethra in incontinent women. An increase in tissue volume (bulking) was observed at the site of BA injection using Micro-CT imaging. The in vivo results demonstrate the host response of BA injections in the rabbit urethra at concentrations of 50 mg/ml and 200 mg/ml. Histological results show there is no morphological difference between BA injected groups and control groups (figure 2). Fluorescent imaging shows BA has infiltrated the urthral tissue and distributed throughout the tissue. Conclusion: BA mimics the structure and properties of natural aggrecan. It shows appropriate host response after injection into the rabbit urethra in vivo and has potential to reduce stiffness and increase the volume of tissue showing promise in reversing deteriorative tissue changes and may thus help incontinent patients. We would like to acknowledge the Coulter Foundation for funding

Biomimetic aggrecan solutions show gel-like rheological behavior

Event Abstract Back to Event Biomimetic aggrecan solutions show gel-like rheological behavior Liam Vesey1, Harsh Patel1, Michele Marcolongo1 and Nicolas Alvarez1 1 Drexel University, Materials Science and Engineering and Chemical and Biological Engineering, United States Several cellular based medicine strategies exist to treat degenerative tissue and restore normal function. Over the past few years, it has been demonstrated that a novel family of biomimetic proteoglycans (BPs) is useful in modulating tissue mechanical properties by molecularly engineering the existing extracellular matrix. Biomimetic proteoglycans mimic the structure and composition of natural proteoglycans through a 3D bottle brush molecular architecture. The BPs of interest are composed of a poly(acrylic) acid core with bristles of chondroitin sulfate (CS) (BA-10-CS, 180kDa). We have observed that the starting CS precursor forms viscous solutions at low concentrations. Similar viscosities are also observed for solutions of synthesized BP. We present a systematic study of the rheological behavior of aqueous solutions of the CS precursor and final BPs as a function of aqueous concentration. We use small amplitude oscillatory shear (SAOS) on a DHR-3 rheometer to elucidate the underlying molecular structure of the bulk aqueous solution. Surprisingly, we find that both CS and BP show viscoelastic behavior typical of a gel and atypical for a solution of short chains. The observed rheological viscoelastic behavior is weakly dependent on CS and BP concentration. Furthermore, very little difference is observed between the CS precursor and the BP molecule. Our results suggest that CS molecules, which are composed of several acrylic acid and primary amine groups capable of hydrogen bonding, are forming a supramolecular network that span the sample volume. This is in agreement with other hydrogen bonding systems found in the literature. The surprising point is that the anchoring of CS molecules onto a poly(acrylic) acid core does not appear to alter the overall supramolecular structure. In addition to SAOS, the molecular structure of the CS aggregates are probed using large amplitude oscillatory shear and steady shear to determine the overall strength of the network and reformation time scales. The rheological study is presented alongside practical injection studies aimed at determining viability for injection of through needles between 16 and 33 gage under a controlled load of 1.8 N (less than thumb pressure to the plunger). Volumetric flow rates were calculated from video images of BP solutions of concentrations between 10 and 200mg/mL. Each solution was able to pass through each needle, regardless of solution concentration, material and needle gage. There was reduced volumetric flow rates for the higher concentrations and lower gage needles. The demonstration of flow through large to very small needles will offer minimally invasive injection strategies to a variety of soft tissues. Coulter Foundation Keywords: Biomimetic, mechanical property, polymer brush, biomacromolecule Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Naturally-derived materials and biopolymers Citation: Vesey L, Patel H, Marcolongo M and Alvarez N (2016). Biomimetic aggrecan solutions show gel-like rheological behavior. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.01381 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Liam Vesey Harsh Patel Michele Marcolongo Nicolas Alvarez Google Liam Vesey Harsh Patel Michele Marcolongo Nicolas Alvarez Google Scholar Liam Vesey Harsh Patel Michele Marcolongo Nicolas Alvarez PubMed Liam Vesey Harsh Patel Michele Marcolongo Nicolas Alvarez Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Editorial introductions

Editorial introductions

PharmaChk: Poor quality medicines screening tool for resource-limited areas

Program/Project Purpose: Substandard medicines account for $75B of a $962B global pharmaceutical market and over 100K preventable deaths annually, leading to tremendous financial loss and emergence of drug resistance. Estimates indicate that 30e50% of all antimalarials are substandard. Additionally, while oxytocin has demonstrated high efficacy in saving maternal lives, it often fails quality tests in LMICs. Similar challenges are seen with many high impact pharmaceuticals. Production and sale of substandard drugs are indicators of a compromised health system that greatly undermine health programs. In absence of proper storage, drugs that initially pass quality testing often deteriorate before the point of sale. This project’s primary objective is to develop a scalable platform that comprehensively screens for substandard medicines, expand implementation from central to regional testing facilities, and create demand through awareness workshops over a four year period. Structure/Method/Design: Project goals: 1) Optimize PharmaChk platform based on pilot user feedback and testing results, 2) Scale-up testing ability to include fixed-dose combination artesunate therapies and oxytocin, 3) Scale-up PharmaChk pilot from one central location to three regional and six community health facilities in Ghana, and 4) Scale-up user demand and awareness by implementing education workshops targeting regulators, pharmacists, manufacturers, and other stakeholders. We work with local stakeholders in Ghana (Procurement and Supplies Department in Ghana’s Ministry of Health and leadership at the Ghanaian FDA and Central Medical Stores) through the US Pharmacopeia’s Promoting Quality of Medicines program. PQM is well-versed with challenges on the ground and provides valuable input on user and technology needs. Outcomes & Evaluation: PharmaChk was pilot tested at the Center for Pharmaceutical Advancement and Training in Accra, Ghana in March 2014. Samples of artesunate pills and injectables were locally sourced and tested using MiniLab , HPLC and PharmaChk. Quantification: Assay accuracy was tested and showed an average error of less than 6% compared to HPLC. Precision/Reliability: Field test revealed excellent device precision, with tests on both tablets and injectables showing< 5% variability. User-friendliness: User training and sample preparation was reduced. Throughput: Testing time was significantly decreased. MiniLab tests took two hours while PharmaChk tests took 15 minutes. Going Forward: Risk mitigation strategy: Inadequate chemical shelf-life might complicate commercial viability of device.User errors and fatigue -we will work closely with users to incorporate their feedback and experiences. Further reduce sample preparation and develop a testing protocol. Basic training protocols need to be designed. Lastly, strong program management, and contingency planning will ensure that we meet our milestones. Funding: US Pharmacopeia, Saving Lives at Birth Consortium, Coulter Foundation, National Collegiate Inventors and Innovators Alliance, Center for Integration of Medicine and Innovative Technology, the National Institutes of Health, USAID. Abstract #: 01ITIS012

Society Awards 2014

Society Awards 2014

Two Years on Linaclotide: Tolerability and Treatment Satisfaction in IBS-C Patients With and Without Diarrhea: Presidential Poster

Introduction: Linaclotide has been shown to improve abdominal pain and stool frequency and was well tolerated in phase 3 clinical trials of IBS-C patients. Here we evaluate the most common LIN adverse event (AE), diarrhea, and assess tolerability and treatment satisfaction (Tx Sat) in IBS-C patients (pts) who rolled over (RO) from a phase 3 trial into a long-term study (LTS) of LIN. Methods: Patients meeting modified Rome II IBS-C criteria were randomized to oral 290 μg LIN or placebo (PBO) qd in a 6-month phase 3 trial. RO pts completed phase 3 and entered an open-label 18-month LTS. Treatment satisfaction (not at all, a little, moderately, quite, very satisfied) and AEs, including any reports of diarrhea, were recorded at all study visits; AE severity was assessed based on pt description. In the LTS, pts could suspend, reduce (145 μg), or withdraw from dosing due to AEs. Treatment satisfaction was assessed in pts with and without diarrhea. Results: Of 535 IBS-C RO pts in the 18-month LTS, 79% had ≥1 AE; 45 pts (8%) withdrew due to an AE. Diarrhea, the most common AE (171 pts; 32%; 0.29/pt-year), was mostly mild or moderate in severity and led to withdrawal in 18 pts (3%) (Table). There were no diarrhea-related SAEs. 131 patients (24%) decreased and/or suspended their LIN dose due to diarrhea; the majority of these patients completed the study (Table). Pts averaged at least moderate Tx Sat; >70% of pts were moderately to very satisfied with LIN treatment regardless of whether they had diarrhea (Figure 1).Figure 1Conclusion: During up to 2 years on LIN, IBS-C pts were moderately to quite satisfied with treatment on average, and LIN was well tolerated. Diarrhea AEs, reported in 32% of IBS-C pts in the 18-month LTS, were generally mild or moderate in severity, easily managed, and infrequently led to study withdrawal. IBS-C pts who reported diarrhea on LIN had similar Tx Sat relative to those who did not report diarrhea. (Studies sponsored by Forest Laboratories, Inc., and Ironwood Pharmaceuticals, Inc.)Table 1: Characterization of Diarrhea in IBS-C Pats on LIN Who Completed a Ph3 Trial and RO Into an LTSDisclosure - William Chey is a paid consultant for Astra Zeneca, Asubio, Astellas, Entera, Furiex, Ironwood, Forest, Merck, Prometheus, Salix, SK, Sunovian, Sucampo, and Takeda, and receives research grants from Coulter Foundation, Ironwood, Nestle, Perrigo, and Prometheus. Steve Shiff is an employee of Forest Research Institute, Inc. and own stock and/or stock options in Forest Laboratories, Inc. Harvey Schneider is a consultant to Forest Research Institute, Inc. Xinming Hao, Bernard Lavins, Jennifer Chickering, Mark Currie, and Jeffrey Johnston Taylor are employees of Ironwood Pharmaceuticals, Inc. and own stock and/or stock options in Ironwood Pharmaceuticals. This research was supported by an industry grant from Forest Research Institute and Ironwood Pharmaceuticals.

Clot-On-A-Chip: A Microfluidic Device To Study Platelet Aggregation and Contractility Under Shear

IntroductionIn primary hemostasis, platelets adhere, activate, and aggregate at the wall of an injured vessel to form a hemostatic plug for the cessation of bleeding. After activation, platelets generate myosin-driven contractile forces to compact the size of the plug in order to reduce the space between platelets and prevent their disaggregation. Hemodynamic shear can be a major effector of platelet function in hemostasis, but its effect on the ability of platelets to produce contractile forces is an open question. Studying the dynamics of platelet aggregation and platelet force generation under hemodynamic shear can provide important insights into hemostasis and thrombosis. MethodWe have developed a microfluidic device that uses microscale blocks to induce platelet aggregation and microscale posts to measure platelet forces in a hemostatic plug. Whole human blood in heparin or citrate is pumped through a microfabricated chip containing microchannels with arrays of blocks and posts arranged along the bottom of a microchannel (Fig. 1). The surface of the blocks and posts are pre-coated with von Willebrand factor and type I collagen to allow for platelet adhesion. As blood is passes over a block, its rectangular shape induces a high shear rate that causes platelets to aggregate on its surface. A flexible micropost is situated behind each block. As platelets aggregate between the block and post, their contractile forces causes the post to bend toward the block. The deflection of the post is recorded under fluorescence microscopy and analyzed using quantitative image analysis of the videos. Since a microscale post bends like a cantilever beam, its deflection can be used to quantify the forces of platelets. ResultsBlebbistatin, a myosin inhibitor, was used to confirm that deflection of the posts by the platelets in heparinized blood was due to myosin activity. When blood was incubated with 2-MeSAMP, a P2Y12 antagonist, platelets were able to aggregate, but their ability to generate contractile forces was substantially reduced. This finding indicates that ADP activation is needed for platelet contractility under shear. The rate of hemodynamic shear was found to influence platelet function, for the rate of platelet aggregation and force generation were found to increase for blood sheared from 2000 to 12,000 s-1. Moreover, platelet aggregation and contractile forces were reduced when glycoprotein Ib-V-IX complex and integrin αIIbβ3 were inhibited with antibody AK2 and antibody fragment c7E3 Fab, respectively. When citrated blood was incubated with tissue plasminogen activator, platelets aggregate and produced contractile forces that increased steadily within the first ten minutes, but then the forces began to subside. ConclusionsOur device can be used to study the role of hemodynamic shear in platelet function and gives insights into the role of platelet forces during hemostasis. Its microscale dimensions also allow us the study the biomechanics involved in the formation of a hemostatic plug during its early stages of growth and stability. [Display omitted] Disclosures:White:Vidacare Corp: Honoraria; Stasys Medical Corp: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; NIH: Research Funding; Coulter Foundation: Research Funding; Washington State Life Sciences Discovery Fund: Research Funding. Sniadecki:Stasys Medical Corporation: Equity Ownership, Founder Other, Membership on an entity's Board of Directors or advisory committees.

Coagulopathy In Trauma Patients Is Accompanied By Oxidation Of a Methionine Residue In The αC Domain Of Fibrinogen and Abnormal Fibrin Polymerization

IntroductionThe concentration of clottable fibrinogen in plasma decreases rapidly after severe traumatic injury and resuscitation, a reduction associated with both greater transfusion requirements and mortality. Fibrinogen is very sensitive to oxidative modification by oxidants generated during ischemia and inflammation, including hypochlorous acid and peroxynitrite, which both can oxidize methionine to methionine sulfoxide. Here, we investigated the potential of this mechanism to contribute to the coagulopathy of trauma. MethodsWe obtained plasma, clinical data, and outcomes from a biorepository of trauma patients presenting to the Emergency Department of a local Level I trauma center. Subjects with plasma INR ≥1.3 were defined as being coagulopathic while those with INR<1.3 were used as non-coagulpathic controls. We precipitated fibrinogen from plasma with glycine and tested it for the presence of methionine sulfoxide using tandem mass spectrometry. We also tested for defects in fibrin polymerization using thrombin time, reptilase time, and fibrinogen activity/antigenic ratio and correlated these changes with the presence of methionine sulfoxide in fibrinogen. The effects of selectively oxidizing certain methionine residues on fibrinogen structure and polymerization were then simulated using molecular dynamics. ResultsTrauma patients who were coagulopathic (N=31) required more blood products in the first 24 hours of care compared to non-coagulopathic subjects (N=30) (mean PRBC: 3.9 vs. 0.5 units, p < 0.001; mean plasma transfused: 3.2 vs. 0.2 units, p < 0.001; and mean platelets transfused: 0.8 vs. 0.2 units, p=0.008) and were more likely to die (29% vs. 10% mortality, ChiSqr Likelihood Ratio p=0.056). Methionine sulfoxide was selectively increased in coagulopathic trauma patients at position 476 in the alpha C domain compared to controls [mean(SEM) 1.8(0.1)% vs. 2.5(0.1)%, p=0.003] and this low-level of oxidation was confirmed to be associated with an increasing reptilase time in vitro. Mean thrombin time (16 vs. 17.8 sec, p = 0.01) and reptilase times (18.2 vs. 23.7 sec, p<0.001) were prolonged in coagulopathic subjects and the fibrinogen activity/antigen ratio, standardized to albumin content to adjust for hemodilution, tended to be higher in coagulopathic patients (1.3 vs. 1.6, p = 0.06). Percent M476alpha oxidation was associated with increased INR (R=0.5, p=0.016), lower clottable fibrinogen concentration (R=-0.546, p=0.009), increased thrombin time (R=0.4, p=0.04), and increased reptilase time (R=0.5, p=0.01). Using multivariate regression, M476alpha oxidation remained significantly associated with reptilase time (M476alpha p=0.035) after adjusting for D-dimer concentration, fibrinogen concentration, and volume of plasma transfused at the time of measurement (whole model R2=0.77, p=0.003). When M476alpha oxidation was introduced into a human fibrinogen alpha C domain homology model, thermodynamic fluctuations were reduced, favoring a more-open configuration for beta sheet-hairpins that are hypothesized to play a role in fibrin monomer lateral aggregation by their dimerization and oligomerization via beta-hairpin swapping. ConclusionsThe level of oxidation of methionine residues in fibrinogen was increased in coagulopathic trauma patients presenting to the Emergency Department. Even at low levels, this modification was associated with altered fibrin polymerization suggesting that it may contribute mechanistically to coagulopathy by altering fibrin alpha C domain dimerization during the lateral aggregation of fibrin monomer. Disclosures:White:Stasys Medical Corp: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Vidacare Corp: Honoraria; NIH: Research Funding; Coulter Foundation: Research Funding; Washington State Life Sciences Discovery Fund: Research Funding.

The Coulter principle: Imaginary origins

In the opening session of CYTO 2013 a spokeswoman for the Wallace H. Coulter Foundation summarized from the podium a story, styled as an urban legend true so far as was known, of Wallace Coulter working toward sizing pigment particles but finding his paint sample frozen. He then supposedly thought of the similar viscosity of paint and blood before first demonstrating the Coulter Principle using his own blood. This requires Wallace being ignorant of the commonly-known disparity between the viscosities of paint and blood, and several listeners subsequently asked me if this story could be true.

MRI-based assessment of lower extremity muscle volumes in patients with knee osteoarthritis

Purpose: To describe lower extremity muscle volumes in patients with knee joint osteoarthritis. Methods: Seven patients with radiographic evidence of knee joint osteoarthritis (6M, 1F, 47±9yrs, 183±5cm, 99±21kg) were scanned on a 3T Siemens Trio MRI scanner. Axial images were acquired for the lower extremity ranging from the ankle to the thoracic vertebre. Thirty-four lower extremity muscles were manually segmented using a semi-automated program written in Matlab. Individual muscle volumes were normalized to patient height and mass. Normalized volumes for each muscle were converted to z-scores relative to a database of volumes collected from normal healthy volunteers. The z-scores were averaged across the patients and means, 95% confidence intervals calculated. Results: On average, the vastus lateralis (z=-4.1, [%CI=4.5,3.8]) rectus femoris (z=-2.2, [-2.6, -1.9]) pectineus (z=-2.5 [-3.0, -1.9]) and obterator externus (z=-2.1 [-2.6, -1.6]) volumes were all more than 2 standard deviations below the corresponding muscle volume means of the healthy group. The vastus intermedius (z=1.4, [0.5, 2.4]) and obterator internus (z=2.0, [0.7, 3.2]) were the only muscles that were on average larger than healthy control with confidence intervals not crossing zero (Fig 1) Conclusions: Patterns of lower extremity muscle deficits include the quadriceps and deep hip muscles that adduct the thigh however, portions of deep hip and thigh musculature may experience an adaptive compensation in patients with chronic knee osteoarthritis. Disclosure: This study is supported by a grant from the Wallis H Coulter Foundation.

Characterization of cell metabolic changes in osteoarthritic labrum cells

Characterization of cell metabolic changes in osteoarthritic labrum cells

Osmotic Pressure of Biomimetic Aggrecan from “Grafting “To Synthesis

IntroductionThe proteoglycan content of the nucleus pulposus (NP) decreases linearly with degeneration.1 This loss of proteoglycan is attributed to both a reduction of the synthesis of aggrecan by the NP cells and an increase degradation by MMP's and proteases, which attack the aggrecan protein core, resulting in aggrecan fragmentation.2-5 We envision a treatment strategy for restoration of the NP, by the addition of an enzymatically-resistant, biomimetic aggrecan (BioAGG) to the NP. This BioAGG, which “grafts” chondroitin sulfate (CS) bristles “to” a synthetic polymer core resistant to enzymatic degradation, will have osmotic pressure as a key functional property for application in the NP. We hypothesize that osmotic pressure can be impacted by both the number and density of the CS bristles incorporated, which will influence the fixed charge density and configurational entropy of this biomimetic macromolecule. Materials and MethodsTo synthesize BioAGG, linear CS molecules (bristles) with primary amine end groups were “grafted to” functional groups along poly-acrylic acid (PAA) chains. Reactions were performed with two PAA molecules at different lengths (250 kDa and 10 kDa) and varying ratios (mol/mol) of CS molecules to AA functional groups (CS; AA). Reaction kinetics was monitored using fluorescamine assay. Resulting macromolecules were purified via membrane dialysis. Polymer samples were dialyzed for 96 hours against DI water (50K MWCO) and lyophilized. Their chemical structure was confirmed with 1H NMR (300 MHz UnityInova NMR Spectrometer, 30 mg/mL samples in D2O).Using a custom membrane osmometer, osmotic pressures of the synthesized molecules were measured and compared to CS alone and an unreacted mixture of PAA and CS. All solutions were tested at the same fixed charge density based on calculations of fixed charge density. ResultsThree different molecules were fabricated using two molecular weights of the PAA core (250 kDa and 10 kDa). For PAA 250-CS samples, we varied the bristle density by controlling the molar ratio of CS to acrylic acid monomer units (CS: AA). PAA250-CS-high with “high” bristle density was synthesized with CS: AA at 1:15, and PAA250-CS-low with “low” bristle density was synthesized with CS:AA at 1:30. The molar ratio of CS: AA for PAA10-CS was kept constant at 1:10. Our theoretical estimation of the molecular weight of BioAGG samples has been done from conjugation data for CS, as determined using a fluorescamine assay, which is sensitive to the reduction in primary amines (PA) on CS. Results of these calculations are seen in Table 1.Table 1 Table 1MoleculeMolecular Wt (kDa)Number of BristlesBristle Spacing (nm)PAA250-CS-high≈2000≈854 to 8PAA250-CS-low≈1100≈4010 to 15PAA250-CS-high≈250≈93 to 5Chemical structures of the synthesized molecules were confirmed via 1H-NMR showing characteristic peaks for CS and corresponding polymer backbones for all purified compounds. No degradation of CS bristles after the synthesis and purification was observed. At the highest concentrations examined (25 mg/mL PAA250: CS) no gelation was observed. Osmotic pressure result of the PAA250-CS-high was 560 kPA about 8× higher than 70 kPA for CS alone (Fig. 1). Interestingly, when the same concentrations of PAA250 and CS are just mixed in solution (PAA250 + CS mixture), the osmotic pressure is close to that of CS alone (∼70 kPA). The same PAA250 with a lower CS bristle density, (PAA250-CS-low) is about 20% lower than the high-density version (410 kPA). A lower molecular weight with high bristle density (PAA10-CS) has an intermediate osmotic pressure between the PAA250-CS and CS alone (140 kPA). ConclusionBioAGG molecules were synthesized using a “grafting to” strategy incorporating CS bristles on PAA polymer backbones. The resultant molecules showed osmotic pressures in excess of those for the fixed charge alone. Moreover, the number and density of CS side chains influences the osmotic pressure supporting a hypothesis that the electrostatic repulsion and conformation entropy derived from the 3D macromolecular bottle-brush structure contributes to the osmotic pressure. BioAGG shows promise for mimicking physical and osmotic properties of natural aggrecan.Acknowledgment Coulter Foundation GrantI confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredOlczyk K. Zeitschrift für Rheumatologie 1994;53:19Roberts S, et.al. Spine 2000;25:3005Goupille P, et.al. Spine 1998;23:1612Le Maitre C, et.al. Biochemical Society Transactions 2007;35:652Kiani C, et al. Cell Research 2002;12:19

Osmotic Pressure of Biomimetic Aggrecan from “Grafting “To Synthesis

Introduction The proteoglycan content of the nucleus pulposus (NP) decreases linearly with degeneration.1 This loss of proteoglycan is attributed to both a reduction of the synthesis of aggrecan by the NP cells and an increase degradation by MMP's and proteases, which attack the aggrecan protein core, resulting in aggrecan fragmentation.2-5 We envision a treatment strategy for restoration of the NP, by the addition of an enzymatically-resistant, biomimetic aggrecan (BioAGG) to the NP. This BioAGG, which “grafts” chondroitin sulfate (CS) bristles “to” a synthetic polymer core resistant to enzymatic degradation, will have osmotic pressure as a key functional property for application in the NP. We hypothesize that osmotic pressure can be impacted by both the number and density of the CS bristles incorporated, which will influence the fixed charge density and configurational entropy of this biomimetic macromolecule. Materials and Methods To synthesize BioAGG, linear CS molecules (bristles) with primary amine end groups were “grafted to” functional groups along poly-acrylic acid (PAA) chains. Reactions were performed with two PAA molecules at different lengths (250 kDa and 10 kDa) and varying ratios (mol/mol) of CS molecules to AA functional groups (CS; AA). Reaction kinetics was monitored using fluorescamine assay. Resulting macromolecules were purified via membrane dialysis. Polymer samples were dialyzed for 96 hours against DI water (50K MWCO) and lyophilized. Their chemical structure was confirmed with 1H NMR (300 MHz UnityInova NMR Spectrometer, 30 mg/mL samples in D2O). Using a custom membrane osmometer, osmotic pressures of the synthesized molecules were measured and compared to CS alone and an unreacted mixture of PAA and CS. All solutions were tested at the same fixed charge density based on calculations of fixed charge density. Results Three different molecules were fabricated using two molecular weights of the PAA core (250 kDa and 10 kDa). For PAA 250-CS samples, we varied the bristle density by controlling the molar ratio of CS to acrylic acid monomer units (CS: AA). PAA250-CS-high with “high” bristle density was synthesized with CS: AA at 1:15, and PAA250-CS-low with “low” bristle density was synthesized with CS:AA at 1:30. The molar ratio of CS: AA for PAA10-CS was kept constant at 1:10. Our theoretical estimation of the molecular weight of BioAGG samples has been done from conjugation data for CS, as determined using a fluorescamine assay, which is sensitive to the reduction in primary amines (PA) on CS. Results of these calculations are seen in Table 1 . [Table: see text] Chemical structures of the synthesized molecules were confirmed via 1H-NMR showing characteristic peaks for CS and corresponding polymer backbones for all purified compounds. No degradation of CS bristles after the synthesis and purification was observed. At the highest concentrations examined (25 mg/mL PAA250: CS) no gelation was observed. Osmotic pressure result of the PAA250-CS-high was 560 kPA about 8× higher than 70 kPA for CS alone (Fig. 1). Interestingly, when the same concentrations of PAA250 and CS are just mixed in solution (PAA250 + CS mixture), the osmotic pressure is close to that of CS alone (∼70 kPA). The same PAA250 with a lower CS bristle density, (PAA250-CS-low) is about 20% lower than the high-density version (410 kPA). A lower molecular weight with high bristle density (PAA10-CS) has an intermediate osmotic pressure between the PAA250-CS and CS alone (140 kPA). Conclusion BioAGG molecules were synthesized using a “grafting to” strategy incorporating CS bristles on PAA polymer backbones. The resultant molecules showed osmotic pressures in excess of those for the fixed charge alone. Moreover, the number and density of CS side chains influences the osmotic pressure supporting a hypothesis that the electrostatic repulsion and conformation entropy derived from the 3D macromolecular bottle-brush structure contributes to the osmotic pressure. BioAGG shows promise for mimicking physical and osmotic properties of natural aggrecan. Acknowledgment Coulter Foundation Grant I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Olczyk K. Zeitschrift für Rheumatologie 1994;53:19 Roberts S, et.al. Spine 2000;25:3005 Goupille P, et.al. Spine 1998;23:1612 Le Maitre C, et.al. Biochemical Society Transactions 2007;35:652 Kiani C, et al. Cell Research 2002;12:19