Delivery of Doxycycline from Plga-coated Hernia Meshes Characterization of Poly(simvastatin)-containing Copolymers and Blends

Statement of Purpose Worldwide, over 20 million hernias are repaired each year, with approximately 700,000 of those occurring in the U.S. alone.1 The high recurrence of hernias, from 32-63%, has been linked to overexpression of matrix metalloproteinases (MMPs).2 Doxycycline inhibits the expression of MMPs and can aid healing of incisional hernias.3,4 The goal of the present studies was to develop coated surgical meshes for local and sustained delivery of doxycycline over a period of two months. Two types of poly(lactic-co-glycolic acid) (PLGA) coatings were examined, and retention of bioactivity was verified. Methods Polypropylene surgical meshes were cut into 2.5 x 2.5 cm squares and dip-coated in a mixture of 10 mL of acetone, 20 mg of doxycycline, and 2 or 5 g of PLGA. Two types of PLGA were tested: 50:50 (ester terminated, IV:0.55-0.75 dL/g, 30-40 kDa) and 75:25 (ester terminated, IV: 0.55-0.75 dL/g). Dip-coatings were repeated, with drying in between, until each dry mesh was 0.35-0.4 g. Coated and dried meshes were placed in 6 mL tubes with 5 mL of phosphate-buffered saline, pH 7.4, (PBS) and gently shaken during incubation at 37°C. At selected time points, meshes were then transferred to new tubes with fresh PBS and the supernatants stored. Supernatants were analyzed using HPLC to quantify the amount of doxycycline released. At selected time points, in a separate experiment, samples were cut from meshes, dried, and analyzed using Kirby-Bauer tests. Staphylococcus aureus were plated on blood-agar plates at a density according to the 0.5 McFarland standard. Meshes were placed on the plates and incubated at 37°C for 24 hours prior to measuring the zones of inhibition. Results 75:25 PLGA coated meshes released doxycycline in a small initial burst and then had a steady release of approximately 20 µg per week for 5 weeks. For the 50:50 PLGA meshes, there was also a small initial burst of doxycycline released within 4 days and then a major release from two weeks until one month. Over the course of 43 days, 62% of the doxycycline loaded into the 50:50 PLGA mesh was recovered, while only 8.5% of the drug was recovered from the 75:25 PLGA coated mesh. This may be due to the greater hydrophobicity and slower degradation of the 75:25 polymer. The Kirby-Bauer tests revealed that the 75:25 PLGA meshes retained their activity, as reflected by antimicrobial properties, over the course of a month. Conclusions Both types of coated …

Abstract: Cancerous tumor cells circulating in the blood were first observed in 18691 and have since become a large focus in cancer research.Circulating tumor cells (CTC's) in the blood are the most common cause of cancer metastasizing to form distant secondary tumors.2Here we review the role of nine commonly studied surface biomarkers that play key roles in cancer progression and metastasis.We then seek to provide quantitative reference data for relative surface marker density between three breast cancer cell lines, two non small cell lung cancer (NSCLC) cell lines and a healthy peripheral blood sample.Surface density of target biomarkers are largely important in the current detection and viable cell isolation methods of CTC's.Epithelial cell adhesion molecule (EpCAM) is the most commonly targeted antigen for targeting cells of epithelial nature in circulation in the blood.However, in most invasive cancers the expression of most key cell adhesion molecules, such as EpCAM and Ecadherin, is known to be down regulated, decreasing the number of binding sites for fluorescent tagging or immobilization and therefore, decreasing the potential of a cell with epithelial nature to be detected.This is consistent with our data of surface expression in comparing the more invasive cell lines, such as MDA-MB-231's to the less invasive cell lines such as MCF-7's and T-47D's.3CTC's are also known to undergo an epithelial to mesenchymal transition (EMT) while in circulation.This transition causes the cell to even further down regulate epithelial specific antigens causing differentiation between normal blood and CTC's to be even more challenging.For future work, we propose an antigen specific fluorescent amplification method that will increase the fluorescent intensity per binding event binding to further distinguish between positive and negative events.Fluorescent polymerization based amplification (PBA) is an antigen specific polymerization technique that coats cells expressing the target antigen in a hydrogel infused with fluorescent nanoparticles.This technique turns one fluorescent event of traditional staining into many fluorescent events through the entanglement fluorescent nanoparticles in the polymer network to amplify the signal associated with one primary binding event.This amplification will allow for a greater separation in fluorescent intensity from auto-fluorescence of cells and non specific staining that is intrinsic to all staining methods.Fluorescent PBA will be compared to the traditional staining immunostaining method using Alexa 488, and an enzymatic fluorescent amplification method, tyramide signal amplification (TSA).Each of these methods of will then be tested using epithelial cells spiked into a healthy blood sample to determine the sensitivity and specificity their ability to positively identify cells of epithelial nature.

Supported by:
The authors acknowledge partial financial support from a National Science Foundation Career award grant awarded to BJB (nsF-1351531)

Abstract:
Cell surface patches provide a potential approach to deliver therapeutics to targeted tissues.Prevailing techniques to manufacture cell surface patches focus on the synthesis of multilayered polymer prior to their attachment to the cells.The application of photolithography on the production of cellular patches has not been fully studied.The goal of this work is to establish an efficient approach to synthesize polymer patches on cell surface via polyethylene (glycol) diacrylate (PEGDA) photopolymerization and photomask-mediated photolithography.PEGDA photo-polymerization triggered by 530nm green light was modeled by Matlab software and experimentally characterized on epoxy microarray, with results showing elevated polymer production with the illuminating duration and light intensity.Furthermore, A549 cell surfaces were biotinylated by sulfo-NHS-LC-biotin or EpCAM primary antibody / biotinylated secondary antibody.PEGDA hydrogels were successfully photopolymerized on biotinylated cell surface.Moreover, photolithographic patterning of PEGDA hydrogel was characterized on an epoxy microarray.PEGDA polymer formation detected by fluorescein signal showed the stronger and shorter illumination had the best chance to accurately generate the desired PEGDA pattern.Finally, photomask-patterned PEGDA polymer production was successfully applied to produce cell surface patches.To sum up, our work first demonstrated the feasibility to pattern the desired size and thickness of cellular patches onto adhesive cells such as cancer cell line or stem cells.Further exploration is necessary to optimize the effects of these patches on cell function, and conjugate therapeutic drug or nanoparticles into these cell patches to commit their therapeutic application.
Supported by: NSF-CBET-1351531 Primary Presenter / email: Wu, P-J / pwu224@g.uky.eduStudent PhD Mentor / e-mail: Berron, B.J. / brad.berron@uky.eduAbstract: Recent studies show that high frequency oscillations (HFOs) can help delineate the epileptogenic zone in individuals with refractory epilepsy.However, HFO incidence could vary with vigilance state, which makes the choice of an appropriate diagnostic baseline for spatiotemporal analysis of HFO activity an important issue.Furthermore, since detection of individual HFOs requires a high data sample rate (> 2000 Hz) and computation on millisecond timescales, a surrogate measure is sought that is easier to compute and correlates with HFO incidence in both spatial and temporal domains.An obvious choice is an estimator of the instantaneous power in the frequency range in which HFOs reside (which we label the HFO index).In this study, we quantify HFO activity and then examine the consequences of vigilance state changes in overnight sleep.We acquired and analyzed ten good quality overnight interictal recordings (1000-2000 Hz sampling rate) from the electrocorticogram (ECoG) in five patients being evaluated for epilepsy surgery.The proportion of epochs containing HFOs varied significantly with vigilance state (Kruskal-Wallis test, p = 0.0055) and also appeared to increase with sleep depth.In addition, there were strong temporal and spatial correlations between HFO counts and HFO index (Spearman's rho of 0.6-0.8 and 0.78-0.88respectively).HFOs show great promise as markers of epileptogenic tissue but analysis of their dynamics is complicated by changes in HFO activity with vigilance state and the lack of uniform guidelines for detecting and labeling them.Our results suggest that simple estimates of HF band power may reflect the relative numbers of HFOs in the ECoG and their variation with vigilance state.
Supported by: EPSCoR RII Track-2 Grant OIA-139068 from the National Science Foundation.Abstract: This study explored the use of hydrostatic pressure (HP) as a mechanobiological parameter to control in vitro endothelial cell (EC) tubulogenesis in 3-D hydrogels as a model microvascular tissue engineering approach.Recently, we showed that HP exposure is a magnitude-dependent stimulus of endothelial tubulogenic sprout formation that may involve VEGF-C/VEGFR-3 autocrine signaling.The present investigation sought to adapt to using endothelial spheroid cultures, which we believe is a more suitable tissue engineering strategy than the Cytodex® beads used in our previous work.At the same time, we also aimed to identify the operating magnitudes and exposure times for HP-sensitive sprout formation as well as verify the involvement of VEGFR-3 signaling.For this purpose, we used a custom-designed system and a 3-D bovine aortic EC spheroid model of sprouting angiogenesis.Using microscopy, we report that an exposure time of 3 days is the minimum duration required to increase EC sprout formation in response to 20 mmHg.Notably, exposure to a 5-mmHg stimulus for 3 days was inhibitory for endothelial spheroid lengths without affect sprout numbers.Moreover, EC spheroids exposed to 40 mmHg also inhibited sprouting activity, but, in this case, by reducing sprout numbers without affecting sprout lengths.Finally, blockade of VEGFR3 signaling abolished the effects of the 20-mmHg HP stimuli on sprout formation.Based on these results, VEGFR-3 dependent EC sprouting appears to exhibit a complex pressure dependence that one may exploit to control microvessel formation.More work, however, is needed to further support and rationalize this possibility.
Supported by: American Heart Association Grant in Aid Abstract: Low back pain (LBP) has been suggested to be a leading cause for chronic health problems in older population.Structure and behavior of tissues constituting musculoskeletal subsystems (responsible for spinal stability) change with aging.Spinal instability and the risk for LBP with aging can indirectly be assessed using measures of viscoelastic behavior of lower back.Passive flexion tests were conducted in upright posture to account for the effects of gravity and corresponding muscle force responses.Viscoelastic response of lower back was characterized using measures of trunk stiffness, viscoelastic relaxation and dissipated energy.The outcome measures were found to be larger in older vs. younger population.Moreover, the trunk stiffness was found to decrease with lower back flexion angle such that the highest trunk stiffness was observed around the neutral posture.Although passive contribution of lower back tissues to trunk stiffness has been reported to be minimal around the neutral posture, the larger trunk stiffness in the neutral posture highlights the important role of active contribution to trunk stiffness and stability.Regarding the larger trunk stiffness in different lower back flexion angles (neutral standing to 40 degrees) in older population, a diminishing contribution of passive and active subsystems to spinal stability is unlikely to be a reason for higher reports of back pain in the elderly.Accordingly, the role of other contributor elements to spinal stability and equilibrium (reflexive response, muscle forces and spinal loads) in increasing the risk for LBP with aging should be further investigated.

April 21, 2016 Lexington Convention Center College of Engineering Biomedical Research Day Poster Presentation Abstracts 145 Abstract Title: Photopolymerization and photolithography of polyethylene glycol-based hydrogels on biotinylated glass and A549 cell surfaces
and a national Cancer institute Cancer nanotechnology traineeship (nCi-CntC) awarded to Jll through grant R25CA153954.

Thursday, April 21, 2016 Lexington Convention Center College of Engineering Biomedical Research Day Poster Presentation Abstracts 146 Abstract Title: Characterization techniques for biological specimens at the UK Electron Microscopy Center
Akbari, A. / azin.akbari@uky.eduStudentPhD Mentor / e-mail: Balk, T.J. / john.balk@uky.eduAbstract:Background: Cerebrovascular disease (CVD) refers to a set of pathological or physiological conditions that affect blood circulation to the brain.CVD is prevalent in the older population.Early detection of CVD in asymptomatic patients is the key for prophylactic interventions that can efficiently prevent stroke.Methods: The study goal is to use MRI with arterial spin labeling (ASL) and T2-fluid attenuation inversion recovery (T2-FLAIR) sequences to quantify cerebral blood flows (CBF) and deep and periventricular white matter hyperintensities (dWMH and pWMH) volumes in elder subjects.Based on Framingham criterion for the estimation of risk to develop CVD, older adults (66 -88 years old) were classified into low-risk (n = 14) and high-risk (n = 12) groups.The correlations between the CBF and neighboring WMH volume in each lobe and over the whole brain are investigated to evaluate the usefulness of these measurements for distinguishing subjects with high or low risk to develop CVD.Results: Mostly, there are high WMH volume in subjects who are classified a high-risk to develop CVD.Total pWMH volume correlated (negatively) with total CBF and with posterior frontal, parietal, temporal and occipital CBF.Notably parietal pWMH volume was correlated with CBF in posterior frontal, parietal temporal and occipital grey-matter.Significant correlations are interpreted to mean that grey-matter CBF decreases with increasing pWMH volume.Discussion and Conclusions: Quantification of WMH and CBF using MRI with multiple functional sequences show potential as biomarkers for the early diagnosis of CVD.The number of subjects maybe is not enough to get a very clear picture for the relation of WMH and CBF with the risk score.Supported by: This study was supported by a pilot award (G.Y.) from the National Institutes of Health (NIH) P30 #AG028383 and the grants from the American Heart Association (AHA) including BGIA No. 2350015 (G.Y.) and Postdoctoral Fellowship Awards No. 11POST7360020 (Y.S.).Bahrani thanks the scholarship support from the Higher Committee of Education Development in Iraq (HCED).Primary Presenter / email: Bahrani, A.A. / ahmed.bahrani@uky.eduStudentPhDMentor / e-mail: Yu, G. / gyu2@uky.eduThursday,

April 21, 2016 Lexington Convention Center College of Engineering Biomedical Research Day Poster Presentation Abstracts 149 Abstract Title: Intraoperative Optical Assessment of Blood Flow Changes in Mastectomy Skin Flaps in Patients with Breast Cancer
Background: The most common complication following prosthesis-based breast reconstruction after removal of breast tumors is mastectomy skin flap necrosis.There are currently no noninvasive methods to assess mastectomy skin flap perfusion.A new portable and inexpensive technology, noncontact near-infrared diffuse correlation spectroscopy (DCS), has been recently developed in our group for noninvasive measurement of tissue blood flow by analyzing the motions of moving red blood cells in deep tissues (~1.5 cm).In this prospective study, we aimed to validate the use of this noncontact device in the prediction of mastectomy skin flap necrosis.Methods: The device was used to continually measure tissue blood flow at three time points: before and immediately after the mastectomy, and after the reconstruction.Flow measurements were done at 2-3 locations and at four depths in each patient along the mastectomy incision.All patients were tracked for the development of complications including skin necrosis and need for further surgery.Results: Eleven patients have been enrolled in the ongoing study.Two patients developed skin necrosis and one of which required re-intervention.The difference in relative blood flow levels after mastectomy in patients who did, and did not develop necrosis was statistically significant, with values of 24.
3% ± 17.7% and 64.1% ± 24.2% of pre-mastectomy baselines (assigning 100%) respectively (p = 0.02, paired t-test).Conclusions: Noncontact DCS is a promising tool that may provide objective information regarding mastectomy skin flap viability intraoperatively, thus allowing surgeons early identification of those compromised and ischemic flaps with the hope of salvaging them.

, April 21, 2016 Lexington Convention Center College of Engineering Biomedical Research Day Poster Presentation Abstracts 150 Abstract Title: Comparison of Post-occlusive Reactive Hyperemia Responses in Thigh Muscles and Tails of Mice Measured by Diffuse Correlation Spectroscopy
Background and Objective: Post-occlusive reactive hyperemia (PORH) protocol is often used to evaluate tissue microvascular function.This study compares blood flow responses in thigh muscles and tails of mice during PORH measured by two custom-designed fiber-optic probes for diffuse correlation spectroscopy (DCS) flow measurements.Methods: We used reflection and transmission probes to measure blood flow responses in thigh muscles and tails of mice (n = 16), sequentially.A 5-min arterial occlusion was created by tying a thin PVC tube around the thigh or tail.Tissue blood flow was continuously measured before, during and after occlusion.Relative changes of blood flow (rBF) were calculated by normalizing time-course data to its preocclusion baseline values.In order to characterize flow recovery after occlusion we quantify the time from the releasing of occlusion to the time that rBF reaches half of its peak value (i.e., T50).Results: We observed similar PORH response trends in both thigh muscles and tails of mice.The mean values of T50 between the thighs (5.38 ± 2.42 s) and tails (20.00 ± 21.87 s) were significantly different (p = 0.01).The linear regression suggested a significant correlation between these two measurements [R2 = 0.30, p = 0.029, T50 (thigh) = 0.079 × T50 (tail) + 4.059].Discussion and Conclusions: Slower reperfusion following the occlusion was observed in the tail as compared to the leg muscle, which is expected due to the smaller amount of vessels in the tail.However, the measurement (probe installation and occlusion procedure) on the tail is much easier than that on the thigh muscle of a mouse.Purpose: To assess off-resonance effects on cardiac strain from spiral cine Displacement Encoding with Stimulated Echoes (DENSE) MRI at 1.5T and 3T Methods: DENSE encodes tissue displacement into phase images, and spatial gradients within the phase images yield cardiac strains, which are valuable measures of cardiac function.When used with long readout durations, spiral acquisitions are prone to distortions and blurring due to off-resonance, which may yield dampened image gradients.Typical spiral cine DENSE acquisitions use 6 spiral interleaves with an 11.1 millisecond readout duration.Five healthy subjects underwent short-axis midventricular 2D spiral cine DENSE at both 3.0 T and 1.5 T. The number of spiral interleaves was varied between 6 and 36 to assess a range of readout durations below 11.1 milliseconds.Magnitude images were visually assessed for blurring and distortions while radial and circumferential strains were quantified from phase images.Strains were correlated against the number of interleaves.Results: At 3.0 T and 1.5 T with the typical, 6-interleaf DENSE acquisition, blurring and distortions were present predominantly in the anterior and lateral left ventricular walls.Those artifacts were markedly reduced in acquisitions with shorter readout durations.Compared to the 36- interleaf acquisition, radial and circumferential strains were underestimated by the 6-interleaf acquisition in those cardiac segments at both field strengths by up to 18.9% and 1.0% (absolute), respectively.Conclusion: Due to off-resonance effects, image quality and measured cardiac strains are dependent on the readout duration of spiral cine DENSE at both 3.0 T and 1.5 T.

Thursday, April 21, 2016 Lexington Convention Center College of Engineering Biomedical Research Day Poster Presentation Abstracts 153 Abstract Title: The Effect of Hypertrophy in CardioCEST Magnetization Transfer Contrast
Introduction: Cardiovascular disease is characterized by both increased interstitial fibrosis and extracellular volume, and by hypertrophic remodeling of individual cardiomyocytes.These structural changes are associated with increased risk of heart failure, arrhythmia, and sudden cardiac death.The endogenous contrast mechanism of magnetization transfer (MT) is influenced by changes in tissue structure, and recently MT-weighted CMR approaches have shown promise in identifying cardiac fibrosis.However, the impact of increased intracellular macromolecule concentration concomitant with hypertrophy on MT contrast remains unknown.In this study we use a murine model of Angiotensin-II (AngII) stimulation to demonstrate that hypertrophy has little effect on MT contrast generated using cardiac chemical exchange saturation transfer (cardioCEST).Methods: Adult male C57Bl/6 mice (n=12) received either constant infusion of AngII (1000ng/kg/min, BACHEM, n = 7) or saline (n = 5) via mini osmotic pump (Alzet).CardioCEST MRI was performed at two saturation frequency offsets (6 and 15 ppm) prior to and 10 days after pump implantation.Maps of the magnetization transfer ratio (MTR) were calculated on a voxel-wise base as MTR(ω)=[(SRef-S(ω))/SRef]*100 and analyzed over the entire myocardium.Following post-treatment scanning, all hearts were harvested, fixed, and stained with picrosirius red and wheatgerm agglutinin.Collagen volume fraction (CVF) and mean cellular cross-sectional area (MCA) were measured using ImageJ (NIH).

, April 21, 2016 Lexington Convention Center College of Engineering Biomedical Research Day Poster Presentation Abstracts 162 Abstract Title: How Aging Affects Viscoelastic Response of the Human Lower Back to Passive Flexion
Reference point indentation (RPI), a form of micro-indentation, offers a novel approach for providing information regarding the mechanical properties of bone.Prior studies using the RPI method have been performed on cortical bone exclusively.RPI is not readily applied to trabecular bone because usable trabecular surface area is limited and it physically alters the substrate.Thus, RPI methods applied to trabecular bone have a single opportunity to effectively utilize available area.The objective of this study was to determine the optimum methodology that efficiently quantifies the resulting RPI parameters.Strain and stress fields produced by RPI were theoretically modeled using finite element analysis.A series of evenly spaced indentations were represented in a 3D model using 3 planes of symmetry.The indentation force was modeled as an axisymmetric non-uniform pressure distribution normal to the indentation surface.It was assumed that the substrate was 280 microns wide and 140 microns deep.Eight ex vivo trabecular bone samples from a homogeneous group of human subjects were indented 9 times (3 trabeculae per sample; 3 indents per trabecula) each using a BioDent RPI device.Variance among: a) bone samples (σb2), b) trabeculae within a bone sample (σt2), and c) indents within a trabecula (σi2) were estimated using standard variance component analysis for balanced hierarchical designs.Each candidate indentation protocol and biopsy sample size was represented by a single, averaged coefficient of variation.The protocol and consequential maximum sample size with the lowest calculated value was selected.The largest factor reducing theoretical variance involved maximizing the number of bone samples.The model showed an experimental indent depth of 50 microns placed at least 175 microns from of a second indent will avoid strain field interaction.Department of Biomedical engineering, U of Kentucky K. Allen-Bryant, College of Nursing, U of Kentucky B. Bazrgari, Department of Biomedical engineering, U of Kentucky