1.

Blood compatibility: Thrombosis and coagulation components (major cells, proteins and biomolecules involved), Virchow's Triad, Vroman Effect, Coagulation Cascade 2. Protein-biomaterial interactions: Physical and Chemical Strageies of Surface Modification, Surface Characterization techniques (qualitative and quantitative) 3. Cell-material interactions (mechanisms involved, characterization strategies) 4. Bioinspired materials 5. Neural physiology, neural inflammation, neural interfacing of biomaterials 7. Cardiovascular devices (artery physiology, role of platelets and endothelial cells, grafts, stents) 8. Drug delivery (ADME, pharmacokinetics, pharmacodynamics, biomaterial application in delivery technology concepts shown in slides, surface modification of particles for intravenous delivery, zero order and higher order release)

1) Blood Compatibility
a) Thrombosis and coagulation components i) Thrombosis = hemostasis in the wrong place pathological state of hemostasis due to thrombus formation ii) Glycocalyx is negatively charged and normally protects against thrombosis iii) Process (i) Endothelial injury vasoconstriction (ii) primary hemostasis (platelet adhesion shape change granule release [ADP, TXA2] recruitment aggregation (hemostatic plug) (iii) Secondary hemostasis (tissue factor release phospholipid complex expression thrombin activation fibrin polymerization) (iv) Thrombus and antithrombotic events (release of 1-PA fibrinolysis; thrombomodulin block the coag cascade, fibrin polymerization, trapped cells) iv) Platelet process. (1) Adhesion: Gp1b vWF, collagen materials? (2) Aggregation: GpIIIb/IIIa fibrinogen (3) Release: many including Thrombin, fibrinogen, ADP v) Factors to know (1) VWF is made in endothelium, all others in liver (2) Intrinsic: Factors XII, XI, IX, VIII, prekallikrein, kiniogen (3) Extrinsic: Factor VII, tissue factor (4) Common: Factors X, V, XIII, fibrinogen, prothrombin

Important: X Xa; Xa turns prothrombin into thrombin, thrombin turns fibrinogen into fibrin. b) Virchows Triad i) Blood changes (hypercoagulable state), flow changes (Stasis), surface changes/injury

BIOMATERIAL SURFACE or

c) Vroman Effect i) Albumin, then fibrinogen, then IgG, finally fXII and HMWK ii) High motility proteins first, which are then replaced by proteins with high affinity d) Coagulation Cascade

e) WHY DOES THROMBOSIS OCCUR ON BIOMATERIALS? i) Flow effects: shear stress, vessel size, Virchows Triad ii) Protein adsorption: major and minor proteins, monolayer v. multilayer, Vroman effect, material surface iii) Platelets and Leukocytes: platelet glycoproteins, integrins/ligands

2) Cardiovascular Biomaterials
a) CV disease is important: major is atherosclerosis b) Blood vessel structure

c) Importance of endothelial cells

d) Glycocalyx i) hydrophilic steric hindrance to protein adsorption ii) 50-100 nm thickmuch thicker than lipid bilayer e) Effects of endothelial cell injury i) Surface adhesion of proteins

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ii) Platelet adhesion and activation thrombus iii) Coagulation cascade activation iv) Complement activation v) Fate of cells downstream (lifespan, embolus) CV Grafts: often used for atherosclerosis a) Most common: peripheral AS (gangrene in legs) b) Hemodialysis shunts in arm vessels c) Coronary AS (myocardial infarction) d) Autologous vessels (bypass grafting, CABG) > synthetic vessels Vascular Grafts a) PET/Dacron. Preferred for large (>4mm) grafts. i) Woven: looped around a thread; not as strong but porous and easier to suture ii) Knitted: filament runs longitudinally and circumferentially. Strong but low porosity. b) PTFE (Teflon) or expanded (ePTFE, Goretex) used in mid-sized and smaller grafts i) Good biocompatibility, durable ii) Hydrophobic, air permeable iii) Low friction coefficient iv) 30 micron diameter porosity c) Crimping: avoid the formation of kinks and bends d) Why do the fail? i) Hyperplasia/restenosis (intimal, anastomotic) ii) Thrombosis iii) Infection iv) BASICALLY: they have no endothelial cells e) Design issues i) Anastomosis: can you suture it? Is the size ok? Is it easy to handle? Stenosis due to intimal hyperplasia? ii) Compliance: shear stress, wave reflections, disturbed flow profiles, stress concentration iii) Biocompatibility: hemolysis, cytotoxic? Leachables? iv) Material properties: surface chemistry, texture, porosity, durability, degradation, fatigue, dilation v) Patient health: anticoagulants, drugs MECHANISM OF THROMBOSIS a) Blood flow protein deposition (fibrinogen) activation of coag cascade (prothrombin, thrombin) platelet activation platelet adhesion thrombus formation embolism b) Fibrinogen mediates interplatelet binding (activate GpIIIb,/IIIa). c) Fibrin is generated and crosslinked via coagulation cascade Surface modification to prevent thrombosis: mimic the clycocalyx a) PEG = random conformation, better at low graft density because it allows whipping around and better coverage. High conformation = entropic effect will dislodge from surface to keep it from stretching b) Saccharide rigid rod = High density

c) Bottle brush: mimic glycocalyx d) Bioactive surfaces can coat with heparin (Carmeda) 7) Stents a) PTCA = percutaneous transluminal coronary angioplasty: put in a balloon to stretch it out b) Coronary Stenting = stents that expand and are left inside the vessel after balloon removed i) Must be flexible, trackable, expandable visible (mechanical) ii) Must be thromboresistant, prevent restenosis, nontoxic (biologic) (1) Drug eluting stents: have a barrier of drug-free polymer & sirolimus in an inner layer. This is released. (2) Stent coatings (a) Biocompatible: carbon, gold, silicon carbide, phosphorylcholine (b) Anticoagulants: heparin, hirudin, Iloprost (c) Corticosteriods: dexamethasone, methylprednisolone (3) Antimitotic agents: (a) PACLITAXEL, SIROLIMUS, angiopeptin, tyrosine kinase inhibitors c) Stent failure due mostly to restenosis and late stent thrombosis (also infection, intimal hyperplasia, acute and subacute thrombosis)

3) Surface Properties of Biomaterials

a) Why? i) Biomaterial surface is the first thing the physiological environment comes in contact with, so it dictates the primary biological response (thrombosis, encapsulation, isolation, degradation) ii) Prevention of certain protein adsorption and cell adhesion, or specific protein adsorption/cell adhesion b) Thermodynamic Methods for Surface Analysis i) Contact angle and Wettability: the angle at which a fluid meets a solid interface (1) More than 90 degrees = hydrophilic (2) Less than 90 degrees = hydrophobic

ii) XPS (ESCA) x-ray photoelectron spectroscopy (1) Release the photoelectrons, can measure kinetic energy and find binding energy (2) USE FOR: polymer surfaces, coatings. (3) Gives you binding energy v. counts/sec (QUANTITATIVE) (4) Non-specific, must dry samples

iii) FTIR/ATR: (1) More info than ESCA but less surface sensitive (2) Use a ray and a detector. Different modes of use. iv) Ellipsometry: indirectly measure adsorbed proteins (non-specific) (1) Linearly polarized light at an angle splits into parallel and perpendicular components which are reflected differently and detected (2) You can measure the film thickness (3) Non-specific, must dry samples v) SURFACE DENSITY (1) SPR, OWLS, QCMB, (XPS, ellipsometry) (a) Kinetic data, lower sensitivity (b) No specificity (2) Direct labeling with radioisotopes (a) High sensitivity, specific. (b) Potential artifacts vi) CONFORMATION/STRUCTURE (1) Scanning probes, spectroscopy (2) AFM, FTIR-ATR, TIRF, CD (a) Low resolution and sensitivity (3) Bio-assay: use antibody as structural probe (a) Specific, sensitive. Shift in affinity = structural changes (but is it the structure or orientation?) (4) ELISA: Enzyme Linked Immunosorbent Assay see protein density v. coating concentration vii) Atomic Force Microscopy (AFM) (1) Similar to a vinyl: have a tip of atoms (4mm diameter at interface, 20-40nm at apex) (2) Good for surface profiling, ultrastructure, surface interaction (3) Output: a picture viii) Light microscopy (1) Micron level resolution (2) Use: cell-material analysis ix) TEM: Transmission Electron Microscopy (1) Electron beam travels through a specimen to make a shadow image (2) 0.5 Angstrom resolution (3) Must be very thin sample x) SEM: Scanning Electron Microscopy (1) Sample surface bombarded with e-, electrons are backscattered (2) 3D image results c) Strategies of modification i) Resist protein adsorption (1) Coat with hydrophilic polymer like PEO (can be shag carpet or bottle brush) ii) Resist specific cell adhesion

(1) Coat with low-affinity protein (e.g. albumin) iii) Promote, enhance specific cell adhesion (1) Optimize surface charge, morphology (roughness) (2) Surface grafting of cell-specific adhesion ligands d) Methods i) Covalent modifications (1) Radiofrequency glow discharge (plasma) (2) Chem and biological grafting (a) Attach PEG or PEO to suppress protein fouling (b) Can also attach it with a biologically active molecule at the other end ii) Non-convalent mods (1) Dip coating from polymer solution (casting) (2) Langmuir-Blodgett Films (a) Dip-coating: like stays with like in terms of hydrophobicity (b) Immersing it into liquid and exploit hydrophilicity/phobicity (c) Usually weak surface adhesion (3) Polymer physisorption and assembly (a) Useful for modifying hydrophobic polymers (b) (4) Self assembled monolayers (SAM) (a) Part of the assembled molecule has high binding affinity for surface substrate molecule (b) Bi-functional: SAM with a biofunctional group at the other end (5) Solution coating must maintain adhesion strength iii) Physiochemical (1) Thinto minimize effect to bulk (2) Simpleable to be produced (3) Resistant to delamination iv) Micro and nano-patterning of polymer surfaces (1) Use a PDMS stamp with ink of SAM, biofunctionals, cell-adhesion molecules

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4) Protein Interactions & Adsorption

a) Adsorption is the first observable event. Influenced by substrate chemistry and surface properties i) Size, hydrophobicity, charge, roughness, stability, unfold rate ii) Cells adhere to PROTEINS NOT MATERIALS (surface proteins cells) b) Vroman effect: protein profile is dynamic at surface proteins replaced by others i) Immediate: proteins in high concentration ii) Later: replaced by proteins with higher affinity

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5) Biomaterial/Cell Interactions
a) Extracellular matrix: ECM i) Structural support for residing cells, and contain cell binding domains ii) Modulate dynamic cellular activities through signal progressions iii) Sequester bioactive molecules iv) Control morphogenesis and wound healing b) Fibrillar Matrix Assembly i) Fibronectin and collagen. Insoluble. Laminin is also a protein ii) Regulate cell adhesion, migration, proliferation, differentiation, ECM co-assembly iii) Cell-mediated process involves self-assembly sites. RGD = integrin-binding motif on proteins

c) Cell adhesion to synthetic surfaces: critical for biomaterials i) Cell-material interactions are mediated by cell receptors that bind to adsorbed proteins. (1) Proteins and surfaces determine inflammatory response, tissue formation, adhesion (2) Passively adsorbed proteins: tend to denature and lose functionality (a) Type, quantity, and structure influenced by substrate (3) Non-fouling, protein resistant bioactive motif (RGD), minimize inflammation d) Surface engineering is important! i) Control cell adhesion surface chemistry (1) Non-fouling and non-adhesive surfaces (2) Surfaces promoting enhanced adsorption (density, activity) (3) Non-adhesive moieties (a) PEG/PEO, phospholipids, carbohydrates (mannitol) ii) Direct cell adhesion biospecific adhesion motifs (RGD) (1) Proteins and peptides. Can functionalize non-adhesive substrates with short adhesive motifs. RGD = adhesion, GYIGSR = adhesion and migration (2) PRO: biospecific, avoid other interactions, synthesis (3) CON: few known motifs, loss of activity compared to ECM iii) Cell growth control: spatial control over presentation of bioactive domains (1) Regulate intercellular interactions; guide cell attachment and proliferation (2) Cell culture environment is important for differentiation/phenotypic responses e) Cell adhesion assays: investigate cell response to biomaterial surfaces i) Wash assay (1) Wash off non-adherent cells. Uneven force. (a) Pro: simple, convenient, widely used (b) Con: not always reproducible, sensitive, limited to short term adhesion ii) Micromanipulation (1) Apply force with micropipette, microprobe, AFM cantilever, laser tweezers (2) Force is obtained from transducer (a) Pro: sensitive, real-time measurements. (b) Con: limited to receptor-ligand binding, need special equipment, single cell measurements iii) Centrifugation (1) Monolayer assembly prewet with PBS FN coating add fluorescently labeled cells and incubate aspirate off non-adherent cells measure fluorescent intensity (2) Apply a centrifugal (normal) force using conventional centrifuge measure fluorescence after and compare (3) Find force from equation (a) Pro: Convenient and simple; population averaged (b) Con: Single applied force per run, low applied force/limited to adhesion iv) Hydrodynamic flow: shear forces due to fluid flow. Proportional force to wall shear stress. Pro: reproducible, controlled, population measurements. Con: flow validation needed, differs based on morphology.

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(1) Spinning disk (2) Radial flow (3) Parallel plate Analysis of Adherent Cell Response i) Cell proliferation: replication and multiplication via mitosis (1) Metabolic activity (redox) (a) Pro: low sample #s (b) Con: non-specific, residual chemicals can alter cell activity (2) DNA Harvest (a) Pro: directly measure cell population (b) Con: need low cell density; multiple time points = high sample volume, need standards (3) Cell count (a) Pro: simple (b) Con: very inaccurate (4) Nucleotide Incorporation (a) Pro: specific to proliferation cells and low sample number (b) Con: time intensive, time specific ii) Protein Expression (1) Real time PCR (a) Pros: sensitive, specific, QUANTITATIVE (b) Cons: standards and primers needed, high cost, time intensive, ***Gene expression does not always mean protein expression (2) Western Blot of Expressed Protein (a) Pros: Sensitive, highly specific (b) Cons: relative values, time, difficulty in sample harvest (amount of protein expressed) (3) Immunofluorescence Imaging (a) Pros: simple, visual image, specific, +++Co-localization of multiple proteins (b) Cons: not quantitative, must bleach sample

6) Neural Electrodes, Neuroinflammation (Lecture 1)

a) General structure

b) Brain tissue composition i) Neurons < 25% ii) Microglia, astrocytes, oligodendrocytes, vascular tissue c) Neural implants disrupt homeostasis and cell populations begin to fluctuate in response. d) Where are barriers present? i) Brain endothelium/blood-brain barrier: isolate the blood from ISF. (1) Barrier is made of cerebrovascular endothelial cells (2) Physical barrier to limit transport through junctions (02 and C02 can go through but large molecules and hydrophilic molecules, drugs are regulated (3) ISF = main component of the extracellular fluid. Contains plasma and transcellular fluid. Provide cells with nutrients and means of waste control. Plasma = blood without blood cells ii) Arachnoid epithelium central layer of the meninges and separates the blood from the subarachnoid CSF. (1) Meninges = membranes enveloping the CNS, purpose is to protect CNS. (a) Outside to inside: Dura mater, arachnoid mater, and pia mater. (2) Subarachnoid = CSF is contained here. Between arachnoid and pia mater. (3) CSF = solution acts as a cushion or buffer for cortex. Provide basic mechanical and immunological protection to the brain. e) Cells of the brain & glial scars i) Microglia: PHAGOCYTOSIS. normally in a quiescent state with short branched processes. 510% of glial pop. (1) Following injury: activation, cell division, migration to injury. Upregulate enzymes, receptors, release inflammatory factors (MHC I,II) (2) Activation persists in injury site for many weeks, until healed. (3) Microglia activation indication of injury and inflammation. ii) Astrocytes: WALLING OFF of antigens.

(1) Resting: round nucleus, 8-10 nm diameter, 30-65% glial pop. Gap junctions w/other astrocytes; maintain neuronal environment. (2) Reactive: large increase in diameter of GFAP filaments, irregular nucleus, proliferation, phagocytosis, migration, secrete ECM proteins, produce neurotrophic and inflammatory cues. iii) Meningeal cells = any injury is rapidly invaded by migrating meningeal cells, which recreate the continuous layer of cells covering the CNS. Also interact with astrocytes to reform the glia limitans that surrounds the CNS. iv) Oligodendrocytes = some die, some survive so glial scars contain some oligodendrogyces and for some time contain myelin debris. Myelin debris is slowly removed by microglia. f) Treating CNS injury and disease: DBS electrodes, injectable hydrogels, electrode arrays, cerebral shunts g) Electrodes lose reliability over time; machine interfaces require proximity to neuronal cell bodies. h) Why does the device fail? i) Implantation causes injury because skull must be exposed and drilled through (1) Infection, hemorrhage, damaged brain barriers, inflammation ii) Inflammatory response causes neuronal loss (1) Insertion of electrode: penetrate BBB and blood vessels. (2) Microglia = first response = activation and try to degrade + engulf electrode (3) Astrocytes encapsulate and wall off electrode, barrier is formed so factors accumulate around the electrode and cause a neurotoxic environment so neurons die around the electrode (4) Neurodegenerative compounds: IL-1, IL-6, TNF-a, NO, RO species, glutamate 7) Neural Electrodes (Lecture 2) a) Process of neurodegeneration

b) What electrode factors involved in neurodegeneration?

Morphology (shape size roughness) porosity, chemistry, anti-inflammatory therapy, modulus, bioinspiration ii) Traditional materials for electrodes (1) Microwires: tungsten, stainless steel, other biocompatible metals (2) Silicon substrate: planar and 3D arrays (3) Ceramic substrate: alumina (a) Advantages of traditional materials: Easy, immediate recording, circuitry (b) Disadvantages: gliosis forms, reduce strength, only record for 1 month (4) Bioinspiration mimic the ECM (5) Methods to reduce glial scarring (bioinspired surfaces) (a) Laminin coating: decreased glial scar, increased cytokine production (b) Resveratrol: reactive oxygen species do not accumulate as much; there is more catalase (i) Reactive Oxygen Species (RO) 1. Oxygen superoxide anion hydrogen peroxide via superoxide dismutase water via catalase (or hydroxyl radical water) (c) Dexamethasone: steroid, anti-inflammatory and immunosuppressant to prevent BBB breakdown (d) Minocycline = neuroprotective antibiotic. Used in conjunction because independently they increase the number of activated microglia. (6) Lattice electrodes increase porosity, decrease inflammation (7) Alginate coating (8) Nanocomposite implants reduce BBB permeability, reduces total microglia/macrophages. Less or no glial scarring but does affect some neuron survival. Still better than solid silicon. (9) Proximity of vessels can also affect the body response. Close to large blood vessels = more pronounced response iii) Bradykinin: cause vasodlation. Upregulates NOS and stimulates pain receptors 8) Drug Delivery a) Pharmacokinetics: Body on the drug (ADME) i) Administration/absorption: crossing of barriers or IV ii) Distribution: by blood or other mechanisms to reach site of action/ Affects concentration at site of action and affects therapeutic action, excretion iii) Metabolism: biotransformation may increase, decrease, or change drug actions iv) Excretion: by urine stool etc. to remove drugs and metabolites v) Engineering: the difference between these two

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vi) eraweraesr b) Pharmacodynamics: the effect of the drug on the body (MOA) c) Drug delivery routes i) Oral/buccal/sublingual (1) PRO: convenient, easy, cheap, painless (2) CON: may cause nausea, stomach irritation, bleeding. Dependent on memory. Firstpass effect may reduce availability (liver) ii) Injection (1) PRO: fast, effective, direct access to circulation (F0 = 100%) (2) CON: unpleasant and painful, may leave scars, most dangerous if dose is off iii) Respiratory/nasal (1) PRO: surface area, lower does, cheap (2) CON: unpleasant and can be painful iv) Rectal (suppository) (1) PRO: partial avoidance of first pass, can give drugs with unpleasant taste (2) CON: irritation v) Controlled release (ENGINEERED)transdermal, implant, injectable targeted etc. (1) PRO: less frequent dosing, targeted action is higher efficacy, controlled therapeutic action (2) CON: complicated and expensive for engineering and validation vi) Engineered delivery: microparticles, nanoparticles, scaffolds vii) Stimuli-responsive materials: hydrogels can undergo volume change when triggered by change in pH, temperature, solvent, ionic strength etc. viii) Reservoir devices (1) Zero order kinetics (2) Use a matrix so the drug is saturated in the core and diffuses out ix) Matrix devices: (1) First order kinetics because the drug is uniformly dispersed (2) Easy to formulate and higher initial release rate than reservoir. x) Diffusion, diffusion and surface degradation, or diffusion and bulk degradation xi) Order of release (1) Zero order does not depend on concentration. Qt = Q0 + K0t (2) First order: drug release depends on concentration. Log(Qt) = Log(Q0) + Kt/2.303 xii) Osmotically controlled can have two layers of drug and a push compartment

xiii) Drug eluting stents xiv) Tiny microneedle array systems to reduce pain xv)