Polymer Properties and Analysis PDF

Topic 2 Polymer: - a structural entity composed of a fixed number of atoms in a given structure that is repeated over and over to form the polymer - Molecular weight - Degree of polymerisation - The number of repeat units in a polymer, indicated by the letter n in the chem...

Topic 2 Polymer: - a structural entity composed of a fixed number of atoms in a given structure that is repeated over and over to form the polymer - Molecular weight - Degree of polymerisation - The number of repeat units in a polymer, indicated by the letter n in the chemical formula for the polymer. - Molecular weight - - Average molecular weight - Number average molecular weight - - Weight average molecular weight  - Polydispersity index - PI = Mw/Mn Types of separating mechanisms Size exclusion chromatography - separation is based on differential partitioning between the mobile and stationary phases - Columns filled with porous beads (10 microm) - Molecules are separated based on the difference in their retention time in the porous stationary phase - Molecule with largest size exits first - Why? Cos it des not enter the porous beads which can impedes its flow - Retention time - e time required for a solute to migrate, or elute, from the column - Instrumentation Steps  - Run polymers of known molecular weight - Plot a calibration curve of MW against standard retention time - Run unknown sample and read out the MW from the retention time Q: A student had a SEC training to measure a polymer sample with narrow, symmetric molecular weight distribution. He made two measurements. In the first measurement, he did the measurement right after preparing the polymer solution.Before the second measurement, he left the polymer solution magnetically stirred in a fume hood without capping the solution vial. Assign the possible SEC curves below to the two measurements and explain the assignments A: the second polymer solution will have a longer retention time. As the solvent will evaporate, thus causing the solution to be lighter, longer retention time Polymer structure Tacticity : polymers with one type of repeat unit Isotactic R groups arranged same side of the chain Syndiotactic The R groups alternate positions on either side of the chain. Atactic The R groups are situated randomly Linearity Linear End-to-end fashion Branched They have chains branching off the main chain. Cross-linked Adjacent chains are joined at certain points via covalent bonds, forming a three dimensional network copolymers : two or more repeating units Random Two mer units are distributed along the chain with no pattern Alternating The mer types alternate along the chain Block Each type of repeat unit is clustered in regions along the chain Graft Homopolymer chains are attached to a different homopolymer main chain Polymer synthesis (polymerisation) Initiation Activation of a monomer through chemical reactions Propogation Monomers continue to successively join the polymer chain and increase its molecular weight. Termination Destruction of the active site: free radical coupling Addition polymerization Initiation propogration  Termination Condensation polymerisaiton - The polymerization occurs through the elimination of one molecule (usually water or acid). Polymerization - The product does not have the same chemical formula as either mer. - Examples of typical polymers are nylons and poly(carbonates) Organization of polymer chains to form macroscopic materials - Percent crystalinity - Polymer chains can fold to form a lamellar structure, which is the basic unit of a polymeric crystal. - Due to the large size and flexibility of polymer chains, polymer materials are rarely 100% crystalline - Polymers are either - semi-crystalline (containing both crystalline and amorphous regions) - multiple intermingled chains (where each has crystalline regions) are located inside the lamellar structures and amorphous regions are found outside - - Amorphous Factors affecting percent crystallinity Anything that prevents chain alignment or discourages secondary bonding between chains will reduce polymer crystallinity Mer side group Large side groups prevent neighboring chains from coming close enough to form a crystalline structure Chain branching Branched polymers have lower percent crystallinity Tacticity Atactic polymers have lower possibility to crystallize than isotactic or syndiotactic polymers Regularity of mer placement in copolymers Alternating and block copolymers have a higher probability of forming crystalline regions than random copolymers Q: Compare the crystallinity of the following polymersA: A: Melting temperature - Melting temperature - RMB THAT ONLY CRYSTALLINE POLYMERS CAN UNDERGO MELTING AT ™ - Below Tm, the crystalline regions of the polymers are highly ordered - Above Tm, the polymer chains show random distribution with no long-range order. - Melting occurs when there are enough atoms or chain segments vibrating to result in overall chain motion. Factors affecting ™ Anything that discourages secondary bonding between chains will decrease Tm Chain branching With more branched chains, molecules are less densely packed, leading to poor secondary bonding between the molecules MW For polymers with the same chemical structure, those with a higher MW will have a higher Tm. Polymers with lower MW have more chain ends. The chain ends can move more freely, as compared to other parts of the polymer molecule. Hence, it needs lesser energy to produce the motion for polymers with smaller MW Glass transition temperature - RMB THAT ONLY ARMORPHOUS POLYMERS POSSESS GLASS TRANSITION TEMPERATURE - Below Tg, the polymer material is glassy and brittle - Above Tg, the chains are mobile enough to produce a rubbery and elastic material - Glass transition occurs when there is enough energy to cause molecular motion around the polymer backbone Thermal transition - Glassy > rubbery > melt - So Tg first then Tm Factors affecting Tg Anything that favors chain vibration and rotation will decrease Tg. Chain flexibility A polymer with more flexible chains requires less energy to achieve the required motion around the backbone and its Tg will be lower Bulky side groupIt reduces the movement around the backbone and increases T MW Polymers with a higher MW will have a higher Tg. DSC for thermal analysis - In DSC, the difference in heat flow into a sample and reference sample is recorded as a function of temperature, while the two are exposed to a controlled temperature ramp. - In heat-flux DSC, the sample and reference are heated from the same heater and the temperature difference between the two cells is measured. - The temperature difference is then converted into heat flow Topic 3 Unit cell - configuration of atoms in a small section of the crystal, that is repeated again and again in three dimensions to form the final material Coordination no: - is the number of nearest-neighbor atoms APF: - Ratio of volume of atoms in a unit cell and total unit cell volume - Crystal structures: Structure Atoms per unit Unit cell volume APF cell FCC Point defects - The creation of defects is favorable because they increase the entropy of the system - Vacancies - There are vacant atomic sites in a structure - Self interstitials - There are “extra” atoms positioned between atomic sites Defects in metal crystal structures: alloys - Substitutional solid solution - Impurity B added to host A leading to a solid solution of B in A - Zn in Mg - rB = rA (1+/0.15) - Interstitial solid solution - C in Fe - rBPGA ** PLGA is a random copolymer so can degrade faster Types of degradation Bulk degradation - The rate of penetration of water into the polymer is greater than the rate at which the polymer is converted into its water-soluble degradation products Surface degradation - The rate of penetration of water into the material is less than the rate of polymer hydrolysis Application of biodegradable PLGA Cranio-maxillofacial implants : anatomy Developmental deformities - An infant's skull is made up of 6 separate cranial bones. - These bones are held together by strong, fibrous, elastic tissues called cranial sutures - For 12-18 months after birth, sutures provide the skull with the flexibility to accommodate brain growth. - After this time, cranial bones fuse together (and remain fused throughout adulthood) Craniosynostosis - the premature fusion of cranial sutures. - Fusion prevents growth perpendicular to the suture and promotes extra growth in the parallel direction to compensate - A distorted skull can increase cranial pressure growth, which can cause vision problems and impede neural development CMF implants - Biodegradable devices are preferred over metal ones in pediatric cases for CMF implants - This is to avoid CMF growth interference in children - There are no intracranial migration, unlike metal implants - Why are they ideal? - Sufficient initial strength for fixation and stabilization - Tailored and predictable degradation concurrent with healing - Biocompatibility: no or low adverse tissue response  - Technically easy to use - Cost-effective Lactosorb as a plating system - PLGA Copolymer for CMF implants - 82% LA - 18% GA - Introduced in 1996 More than 50,000 successful implantations - How to use? - After the bones have been repositioned to correct the craniofacial defect, sheets of LactoSorb are screwed into the bone to secure and support bone fragments CMF fixation surgeries - Surgeons correct defects of the face or skull by cutting apart the skull and strategically piecing it back together - Overview of procedure - Skin is cut and moved to exposed bone - Osteotomy : surgical cutting of the bone - Bone rearranged to correct defect - Fixation device put in place - Skin closed Degradation of lactosorb device Controlled drug delivery - Normally, the drug concentration in blood rapidly rises and then exponentially decays as the drug is metabolized and/or eliminated from the body, resulting in transient therapeutic window. - Drug concentrations above the toxic level lead to side effects, and those below the minimum effective level are not therapeutically effective. - Controlled release formulations can maintain a desired concentration for long periods of time without reaching the toxic level or dropping below the effective level.  - Hydrogel properties and application What are they? - Hydrogels are water-swollen, crosslinked polymeric structures. - The polymer chains in hydrogel can be crosslinked by covalent bonds, physical entanglements, secondary interactions, or crystallites bringing together two or more polymer chains Why good biomaterials? - high water content and biocompatibility Applications in biomed - Surgical sealant (thin film) - Tissue engineering (permeable for nutrients) - Drug delivery (pores for drug encapsulation) - Medical and biological sensors (stimuli-responsive hydrogels Hydrogel as a surgical sealant How and why? - A sealant can seal air, fluid and blood leaks. - Synthetic hydrogel mechanically bonds to the tissue and allows healing underneath. Contrary to a hemostat,** a sealant does not require the time for blood to clot and the strength of the blood clot to form. - A sealant can be applied pro-actively to a dry field. It does not need blood to react and creates a strong mechanical bond with the underlying tissue, sealing blood, fluids and air leaks Applications - Prevent gas leaks in lung - Prevent blood leaks from vessel - Prevent cerebrospinal fluid leakage - Serve as local drug-delivery depot Chem reaction to form hydrogel Why hydrogels as a - Superior strength for longer time periods surgical sealant - Minimal swelling - Prevent adhesions - Biodegradable How to prevent CSF - CSF leakage can result in leakage? - Headache - Vomiting - Dizziness - Meningitis - Arachnoiditis - Pseudomeningocele formation - Hydrogel sealant stops CSF leakage caused by surgically induced dural incisions and dural tears, thereby eliminating complications associated with CSF leaks Topic 6 Why do we use hydroxyapatite? - Same chem composition as bone - Has good compatibility with human bone tissue - Will encourage osseointegration - Coated using plasma spraying What is the function of osseointegration - Fast mineralisation of the surface - Stable binding between the mineral phase and the implant surface - Structural continuity to the surrounding bone Strategies for osseointegration - Increase surface roughness - Macroscale surface features - Microscale surface features - Hydroxyapatite surface coatings Why not use peptides to facilitate cell adhesion - No because restenosis will occur - Whereby the blood vessels will narrow - Peptides will cause cell adhesion, which will narrow the blood vessels Drug eluting stent vs bare metal stent - Drug eluting stent will slowly release drug through diffusion to block cell proliferation and prevent fibrosis Design for biodegradable metal stent - - Mg allows - Corrosion in physiological conditions - Can be used to develop biodegradable stents Zinc is able to form alloys with magnesium when the Zinc weight is up to 4.0% of the magnesium weight. Beyond this weight limit, Zinc forms a separate phase in the Zinc-Magnesium system. Discuss how the anti corrosion property of the materials changes when the weight of Zinc increases from 2.0% to 6%. - At 2%, Zn and Mg form an alloy (solid solution) together. Because Zn has a greater standard electrode potential, the alloy is less reactive than Mg. However, when Zinc forms a separate phase at 6%, the more reactive Mg will become the metal that undergoes cathodic protection of Zn. Therefore 2% Zn sample is more stable Topic 7 Secondary protein - H bonds between the same polypeptide chain at different locations between the peptide bonds - Alpha helix - Hydrogen bonds between groups on the backbones; different amides but same pp fragment - Intra-fragment bonding between residues in a single chain - Beta pleated sheet - Hydrogen bonds between 2 amide groups on the backbones of 2 different peptide fragment - Inter-fragment bonding between 2 fragments Tertiary structure - Interactions between R groups in 1 peptide chain Quaternary structure - 3D arrangement of these subunits (polypeptides) - Interactions between the R groups of the amino acids on different pp chains Hydrophilic and charged - Positive - NH3+ or NH2+ - Negative - O- - For arginine, the double bonded NH carries a positive charge  - For histidine, a bit special haha - Hydrophilic but uncharged - OH, NH2, Hydrophobic - Tyrosine is hydrophobic cos of its bulky benzene group Special a.a. - Cysteine - Selenocysteiene - Glycine - Proline - Fewer degrees of rotational freedown than other amino acids (limit protein folding in 3D) Isoelectric point - The pH at which a molecule or surface carries no net electrical charge - pHpI, negative Cysteine is used for hair treatment as the cross-linking can keep protein in a certain shape Factors dictating protein adsorption - Dehydration of surface protein (hydrophobicity) - Aggregate in solution to reduce exposure to H2O - To reduce free energy - Which will lead to dehydration  - After aggregation, some H2O will be freed up to reduce free energy - Free up some ordered water, S will increase - Hydrophobic interaction important in - Micelles and lipid bilayer - Allow protein to fold - Membrane proteins insert into non-polar lipid environment - Protein small molecule interactions - Charged - Different parts of protein got different surface charge - So part of the protein will interact with the surface - If the biomaterial has positive charges, the parts of the protein carrying the negative charge will interact with the surface and vice versa - Structural rearrangement - Protein with more intermolecular -cross linking , more stable - Will interact with biomaterial surface less - If the intermolecular bonds are small, protein will unfold and lead to primary structure for adsorption (more cos surface area contact is more) - Less stable - Unfold greater extent and more contact points, leading to greater adsorption on the biomaterial surface - Higher unfolding rate - Form surface contacts more easily Reversibility of protein adsorption - Changing from reversible to irreversible - Protein adsorp knot surface with weak interactions (hydrophobic and electrostatic interactions) - Protein undergo conformational changes and have more binding sites Properties that affect surface interaction - Size - More sites for surface contact - Charge - Hydrophobicity - More hydrophobic, adsorb more readily on surface - Interaction: - Water-water hydrogen bonds are broken to create a cavity for the solute molecules - The solute molecules are entrapped in a structure formed by highly ordered water molecules that is held tgt by hydrogen bonds  - Hydrophobic interactions cause the molecules to come tgt to reduce overall hydrophobic surface area exposed which leads to dehydration and some ordered water is freed - Structure - Less stable - Lesser intramolecular crosslinking, less table, can unfold greater - Unfold greater extent and more contact points - Higher unfolding rate - Form surface contacts more easily Physisorption - Weak interaction Chemisorption - Formation of covalent bonds Reversibility of protein adsorption - From Physisorption to Chemisorption - Residence time increase, then conformational change, then permanent binding Langmuir model - Adsorption rate - r=ka[P][S] - Desorption - R =kd[PS] - Hypothesis on adsorption kinetics What happens in a multicomponent protein solution Transient competitive adsorption - Smaller and more conc. Protein adsorp on surface first - Larger proteins with higher affinity to the surface arrive later - Push smaller protein away due to their stronger affinity - Smaller proteins will slowly be displaced by larger proteins with stronger interacting effect Vroman effect - proteins with higher conc will arrive and adsorp first but eventually displaced by those with greater surface affinity - Order Of absorb - Albumin - igG - Fibrinogen - Fibronectin - High MW kininogen Affinity liquid chromatography - Separate liquid based on stationary and mobile phase - Based on affinity of solute and active sites of stationary phase - Mobile phase - Liquid solvent (non-polar) - Non-polar: hexane - Polar (reverse): water - Stationary phase - Small silica/polymer beads (polar) - Non-polar (reverse): C18 - Retention tip - Weakest affinity exit first - Reverse phase : opposite Colorimetric assays - Colour change due to the presence of proteins - Chromophore which absorbs UV light - A = ElC - A absorption intensity - C concentration - L width - E constant Bradford assay (a type of colorimetric assay) - Measure protein concentration - Detection limit is 1-20 microg/ml and 100-1500 microg.ml - Based on the absorbance maximum of acidic Coomassie Brilliant Blue G-250 which shifts from 465nm to 595nm when protein binding occur - The dye is Through van der waals reaction with the carboxyl groups - The dye through electrostatic interaction with the amino group - The dye exists in 3 forms: anionic (blue), cationic (red), neutral (green) - The red dye reacts with protein to turn blue - Advantages - Not affected by sucrose / carbs - Not affected by Na+ and K+ ions - Disadvantages - Affected by detergents such as SDS, Triton X100 - Can non-specifically bind with amino acid - ELISA - Antibody attached to the bottom of the well - This specific antibody is designed to bind to a protein of interest - Another enzyme-linked antibody binds to the protein - Adding the substrate to the enzyme converts to substrate t another colour - Types: Direct Indirect Sandwich  - What is the secondary antibody - HRP (horseradish peroxidase) - Enzyme that catalyzes the oxidation reaction of substrate to generate color changes (detected by UV-vis spectrometer - - Quantification based on colour change - Protein amount proportional to degree of colour change - To find specific protein and determine its concentration - Can als be fluorescently labeled or radio-labeled (detect specific protein and determine is concentration/amount Fluorescence - - Emission of light by a substance that has absorbed light - Reorganization of the solvent around the molecular structure - Absorption (excitation) have higher energy (shorter wavelength) - Fluorescence (emission) have lower energy (longer wavelength) Western blotting - Using antibodies to identify proteins - SDS-PAGE - Separation of protein sample by size. The sample is treated wit SDS and causes them to unfold - Protein transfer - Protein transferred to - PVDF (for hydrophobic) - or nitrocellulose membrane (for hydrophilic) - Via electric current - Same procedure like ELISA - Unlike ELISA, not sandwiched this time - Colour change detected by camera or other means - More detailed explanation: - Upon treatment with SDS, the proteins in the sample are denatured and covered by negative charges - The proteins, withs imalr mass to charge ratio, travel to positive charge anodes and are separated based on their molecular weight - After finding protein sample with that molecular weight, we use western blot to determine the protein of interest The gel is placed on top of nitrocellulose membrane, which is capable to bind to large amounts of protein. The proteins are transferred from the gel onto the membrane Gel mrmbane construct flanked by filter papers. They are soaked in buffer to facilitate the electricity transmission Everything fixed between cathode and anode The electrode transfers the negative charged protein from the gel to the membrane The membrane is then incubated several times with BSA which blocks and prevent primary antibody from unspecifically binding to the membrane or to other proteins Membrane is then incubated with primary antibody which binds with protein of interest Then incubation with secondary antibody  Detection with horse radish peroxidancse This then causes the band to fluorescent Comparison Topic 8 Cell membrane - Channel proteins - Diffusion of ions - Carrier proteins - For active transport - Receptors - To respond to the environment Cytoskeleton - Cellular division (microtubules) - Intracellular transport - Cell motion - Pseudopodia in response to external stimuli or during locomotion - Maintains cell shape - Types - Actin - Contractile apparatus - ​cell movement - intra-cell motile processes - e.g. movement of vesicles, phagocytosis, cytokinesis - Intermediate filament - Attach to receptor and translate external signals - ​Provides mechanical strength to cells - Cell shape, cell adhesion, cellular movement, division - Provide structural reinforcement of the cell membrane between cell and ECM - Don’t give any cell motility and contractility - Connecting at cell junctions called desmosomes - Without them, stretching or compressing will cause breakage - Microtubule - Separation of DNA before cell division - Structural support - Intracellular transport - Axonal transport - Vesicles with proteins move along microtubule of axon - Cell organization - Large in diameter, helps in cell organization Transcription - RNA polymerase synthesizes the linear mRNA strands of the complementary DNA Translation - Production of proteins by decoding mRNA produced in transcription Nuclear envelope - Separate the nucleus Nuclear pore - Allos only specific molecules to enter or exit the nucleus Membrane receptors and cell contacts - Cell-cell contacts - Tight junctions - (due to transmembrane proteins claudin and occludin) - Form tight seal to prevent substances from lateral diffusion - They are most common in epithelia - Sheets of cells that provide interface between masses of cells and a cavity/space (a lumen) - Multiprotein junctional complex - Membranes join tgt forming an impermeable barrier to fluid - They composed of a branching network of sealing strands - These include the distal convoluted tubule, collecting duct of nephron in the kidney, bile ducts ramifying through liver tissues - Hold cells together  - Limit passage of molecules and ions between cells - Block movement of the integral membrane proteins between the apical and basal membrane - Gap junctions - Small hydrophilic channels (1.5-2 nm) that permit free transportation of ions and molecules which alters cytoskeleton -> alter gene - Receptor of growth factors -> cascade of intracellular factors -> alters gene - Change in gene expression affects: - Communication - Protein synthesis - Adhesion, migration and spreading - Viability and survival - Proliferation - Differentiation Cell death Necrosis vs apoptosis Necrosis Apoptosis ○ Not planned ○ Programmed ○ Inflammatory ○ Non-inflammatory ○ Always pathological ○ Sometimes pathological ○ Macrophage digestion ○ Mitochondrial factor ○ Cell membrane release destroyed ○ Cell membrane intact ○ Expired cells are ○ Specific binding on phagocytosed by membrane that directs inflammatory cells cell death Cell proliferation - Labile (unstable) cells: replicate continuously - Permanent cells: terminally differentiated and lost the ability to divide - Stable cells: somewhere between labile and permanent. After one division, they take on a specific function Cell motion - Cell adhesion - Cell surface binding through non-covalent interactions and ligand receptor interaction - Spreading - Pseudopodia extend for post attachment - Interaction between integrin receptors and ligands - Migration - Pseudopodia adheres to surface - Generate contractile force and release rear receptors which causes forward motion Cell quantification : MTT assay - Colorimetric assay to measure cel metabolic activity - Enzyme reduce MTT to formazan (yellow to purple) - Can determine cytotoxicity of drugs on cells - - Only in viable cells, we have mitochondrial reductase - Steps - Culture the cells - Plate the cells to be assessed 24 hours before the assay - Add MTT to the cells - Since MTT is light sensitive, make sure not exposed - Incubate for 4 hours - Add DMSO then incubate for another 2 hours - Standard curve of absorbance against cell number - Control and treatment undergoing MTT assay Assays - Cytotoxicity - Direct contact assay - For those that can directly dissolve in culture media - Put cells in culture dish and media - Test material placed on to of cell - After 1 day, cells in area underneath and around sample observed  - Evaluate on swelling and cell lysis to determine if got any cell loss - Qualitative analysis - Compare cytotoxicity with positive and negative controls using microscope - Quantitative analysis - MTT measure cell metabolic activity - Cell viability = no. of treated cells / no. of control cells - Agar diffusion test - Similar to direct contact, except cells are covered with agar before contact to test material - After 1-3 days of exposure then test - The greater the no. of affected zones or farther away from specimen, then greater cytotoxicity - - - Elution assay - To test if the degradable substance from the biomaterial is toxic or not - Test cytotoxicty of leachable substances from biomat - Test biomaterial is placed in fresh media for 24 hrs - Cells are plated into well plates and allowed to adhere for 24 hrs - Media containing soluble products diffused from biomaterial is transferred into culture well plates - Cell motion - Adhesion assay - To see if cells can adhere to biomat surface - Let cells adhere and interact with substrate for certain period of time - Remove the no adhere cells - Quantify no. of cells using MTT assay  - Migration assay - - - https://www.merckmillipore.com/SG/en/life-science-research/antibodies-a ssays/assays-overview/cell-invasion-migration-assays/boyden-chamber-t echnique/I0qb.qB.KSMAAAFANtY.1ZcQ,nav?ReferrerURL=https%3A%2F %2Fwww.bing.com%2F&bd=1 - Gene and protein (Interrogate gene expression and cell function) - DNA: Polymerase Chain Reaction (PCR) (find old notes) - for rapid production of a very large number of copies of a particular fragment of DNA from a small quantity of DNA  - - At 95C, - Denaturation to break the H bond between base pairs of the 2 DNA strand - Separate the 2 DNA strands to form 2 single stranded DNA, - leaving bases exposed - At 54C, - Annealing take place - Primers will form H bond with the bases of the single stranded DNA - dNTP will form H bonds with the complementary bases of the single stranded DNA - At 72C, - Extension - DNA polymerase will attach to primer - DNA polymerase can only be attached to a double stranded DNA so we need a primer for DNA polymerase - Move along the the free nucleotides complementary to the template strand to form phosphodiester bonds between the nucleotides - Builds new DNA strands - Lets say this is done in 30 cycles - So the formula to calculate is 2^n, n is the number of cycle - - - mRNA : Reverse transcription PCR (RT-PCR) - Southern (DNA) and Northern blotting (mRNA) - Similar technique to Western blotting - No SDS is added since DNA is already negatively charged - Detection probe is either a fluorescent molecule/radioisotope  - Detailed explanation of southern blotting - Genomic DNA treated with restriction enzyme - Fragments are segregated through agarose gel electrophoresis - Agarose treated with NaOH to separate double stranded DNA from single stranded - Ss DNA transferred to a membrane by placing the membrane on top of the agarose gel, then using layers of filter paper on top to press it down (transfer occur through capillary action) - Once transfer complete, the membrane is heated in hot air oven or exposed to UV light to make sure DNA permanently attached with the membrane - Probe has nucleotide sequence complimentary to desired DNA fragment (labelled with fluorescence dye) - X ray film is placed on top of the membrane, which shows presence or absence of gene  - Protein assys - Immunostaining - Immunohistochemistry (IHC) detects antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues - Primary antibody is used to target antigens. - It is used to identify specific proteins (antigens). - It can be visualized via a microscope - Very similar to ELISA - Can be used in this manner - After biomaterials implantation for a certain period of time, tissue surrounding the biomaterial is harvested - Fluorescence labeled antibody that has specific binding with the receptor (aka antigen) on the surface of neutrophils or macrophages is used to stain the tissue - Fluorescence microscopy is used to detect the fluorescence signal and and quantify number of neutrophils or macrophages around biomaterials, indicative of acute inflammation - - Similarity and difference between ELISA and immunostaining https://pediaa.com/what-is-the-difference-between-elisa-and-immunofluorescence-assay/ similarities : - ELISA and IFA rely on the specific binding of antibodies to their corresponding antigens. - Both assays are highly sensitive. This helps in the detection of low concentrations of antigens or antibodies in a sample. - Questions : - How to test cytotoxicity for degradable scaffold for tissue engineering - Direct contact assay to test for cytotoxicity of scaffold - Elution assay to test potential cytotoxicity of the degraded components - MTT to quantify cell numbers - You decided to use a tissue engineering approach where a scaffold with no cells is implanted, and the surrounding cells will infiltrate the implant and start the regenerative process. To aid cell infiltration, a chemical agent will be loaded into the scaffold and released upon implantation to attract neighboring cells. How would you determine whether this chemical agent has the desired effect on an in vitro test - Usually for cells infiltrating etc (we want to test the ability of the cells to migrate across the polycarbonate membrane through the presence of the chem agent Boyden chamber assay - The cells are placed in the upper inner chamber and the chemical agent is placed in the bottom chamber Questions for quiz II Topic 9 Overview of immune response Innate immune response (non-specific) Acquired immune response (specific) - Recruitment of immune cells to sites - Recognition of specific antigen of infection through the production of - Specific responses maximized to chemokines and cytokines eliminate specific pathogens/infected - Activation of complement cascade to cells identify bacteria, activate cells and - Development of immunological promote clearance memory, i.e., vaccination - Identification/removal of foreign - Slow response (3-5 days) substances - Immediate response Leukocytes Leukocytes Types of innate defenses - Anatomic barriers - Skin, tight junction and mucous membranes - Physiological barriers - Temperature of body, low pH in stomach - Inflammation - Acute inflammation is the initial response of the body to harmful stimuli.  - And, it is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. Role of neutrophil - A key portion of the defense mechanism presented by inflammatory response is the influx of phagocytic cells - Neutrophils first bind to vascular endothelium, then penetrate the endothelial layer lining of the blood vessel, and finally migrate to the area of inflammation. - - Read more from my old notes Actions of neutrophils - Phagocytosis - Main function - Granules of bactericidal agents and enzymes - Secretion of chemical mediators (cytokines) - Interleukin (IL-8) : attracts neutrophils - Macrophage inflammatory protein (MIP)-1a: attracts monocytes - These chemoattractants are released by the foreign substances (aka bacteria) - Respiratory burst - Rapid release of reactive oxygen and nitrogen species - Another means to kill foreign organisms - May cause unwanted tissue damage Roles of monocytes and macrophages - Inflammatory response - In 1 hour, the neutrophil will arrive - 5-6 hrs later, monocytes arrive at the site of damage - Monocyte enlarge to become macrophages - The macrophage mature for 8 hours - Swelling of the cell and formation of large quantity of lysosome - What does the monocytes do - Phagocytosis and secretion of chem mediators  - they are longer lived and have greater phagocytic ability - Role as antigen presenting cell - Direct connection between the innate and acquired immune responses problematic biomaterials - Do not degrade - the material will stay in macrophage till it dies. - Lead to repeated recruitment of the macrophage - Clinical condition will develop, - Silicosis (where silica inhaled into lungs and fibrous tissue) Termination of acute inflammation - Purpose of Acute Inflammation - Recover from injury and prevent the invasion of foreign organisms. - Why terminate - Some physiological changes associated with inflammation is detrimental to tissue function if they are wide spread or last for too long - How is it done - Chemical mediators that provide system checks and balances to ensure localized effects. E.g., the production of IL-1 receptor agonist (IL-1ra). - The production of substances that limit inflammatory response Complement system - A family of proteins that serve many roles in both the innate and acquired immune response - Basic functions - Elimination of foreign elements - Promotes phagocytosis of antigens - Triggers inflammation and other immunoregulatory molecules - Immune clearance by forming membrane attack complex 3 pathways activate the complement system Membrane attack complex Regulation of complement system - Why regulation - Confine its effects to a localized area and prevent host cell from being lysed by mechanisms intended to kill foreign pathogen - Enzymatic control - The short half-life of certain enzymes regulates various activities in the complement system - Regulatory proteins - Regulator of complement activation (RCA) proteins compete for key binding sites, and block the activation. - Decay-accelerating factor (DAF) binds to enzyme and forces dissociation of the enzymatic component from the cell-bound component Assays for innate immune response - Cell types commonly used are - Human blood cells - Endothelial cells - Leukocytes assays mostly using neutrophils or macrophages to test for cell activation - Cell adhesion and spreading - Cell death - Migration - Cytokine release - Cell surface marker expression Flow cytometry 1) Known as a laser-based technology widely used to phenotype cells using antibodies directed against surface markers a) Cells are tagged with antibodies through the antigens b) Need to choose antibody that wil bind specifically to the cell u want to test c) Antibody will be directly tagged with a fluorophore d) Can do internally fluorescence tagging i) Permeabelising ii) So antibodies can move through the holes in the membrane and find their target on the inside of the cell 2) Cells are fluorescently tagged 3) Injected in a pressurized sheath fluid a) To make sure that they move at a constant rate through the rest of the machine 4) Interrogated via laser 5) Fluorescence at various wavelengths detected enable quantification of the relative amounts of fluorescent and non-fluorescent cells a) Forward scatter of the laser i) Tells us about cell size (larger than higher) b) Side scatter i) Granularity (1) Cells that are more stuffed inside cytoplasm 6) FACS (Flow Activated Cell Sorting) a) Separate different cell components i) Laser excitation causes cells to pose electrical charge proportion to the fluoresnece intensity and thus cells will be deflected  ii) Other assays - Response of endothelial cells - Testing for up-regulation of specific cell surface receptors or ligands - Cell surface markers can be analyzed using FACS - Model systems from tissue engineering (TE) constructs - Co-cultures of TE skin replacement with biomaterial of interest - Monitor the production of cytokines measured using ELISA Acquired immune response - is mediated by lymphocytes circulating constantly in the blood and tissues. - Main function - Antigen presentation and recognition - Responses to maximally eliminate specific pathogens - Derivation of immunological memory - Main characteristics - Specificity (response to antigen) - Diversity - self/non-self recognition - Immunologic memory Type of acquired immunity Humoral immunity Cellular immunity Actions of antibodies against foreign Utilize specialized lymphocytes (T cells) to substance such as bacteria detect altered self cells Main definitions antigen : substance that bind specifically to antibodies/T cell receptors Antibody (immunoglobulin glycoprotein): a protein which identify and nuetralises foreign substances T cell receptors (TCR): Molecule on T cells that recognises antigens bound to MHC proteins Hapten: low MW molecule that combines with high MW molecules to produce a greater immune repsonse Lymphocyte - B cell - Mediators of humoral immunity and produce specific antibodies - T cell - Tc and Th involved in cellular immunity and produce proteins with high specificity (TCR) B cell T cell What are they Mediators of humoral immunity and Tc and Th involved in cellular produce specific antibodies immunity and produce proteins with high specificity (TCR) Cell maturation B cells are formed and processed - T cells are formed in the bone in the bone marrow but mature in marrow but mature in the the peripheral lymphoid tissues thymus. (lymph node and spleen). - T cells become activated in response to antigen-MHC complexes Role - Make antibodies T helper cells - Act as APC and develop - Expresses CD4 glycoproteins  into B cells - Recognise MHC class II molecules Upon activation, - Activated T cells form a - Proliferate to form memory colonal Th effector and Th cells and plasma cells memory cell (effector cells) - Effector secrete cytokines - Memory cells: - Stimulate B cell - Express membrane growth and bound antibodies stimulation - Plasma cells produce - Stimulate proliferation soluble antibodies of Th cell - Plasma cells can - Promote chemotaxsi release up to 2,000 and macroohage antibodies per activation second at maturity - Stimulation of Tc (cytotoxic cells) Tc (cytotoxic T cells) - Expresses CD8 glycoproteins - Recognizes MHC Class I molecules - Effetor T cell secrete perforins which lyses cells - To prevent infection and cancer What are - Multimeric glycoproteins antibodies - Expressed in both membrane-bound and soluble forms - At surface protein - immunoglobulin receptor plays an important role in activation of B cells. - As soluble molecules - they are found in the blood and secretions, serving to recognize and clear foreign antigens from the circulation. Got great structural diversity Structure of - 4 pp chains antibodies - Heavy chains (55-70 kDA) - Light chains (24 kDA)  - Linked via disulfide bonds - Cleft for antigen bonding is that variable portion (Fab) - Remaining part is constant portion (Fc) Classes of antibodies Mechanism of - Agglutination antibody actions - Clumping of particles - Lysis  - Direct attack on cell membrane - Precipitation - Enlargement of antibody-antigen complexes till it becomes insoluble - Neutralisaiton - Antibodies bind and cover active/toxic sites Steps in acquired immune response Recognition of antigen MHC molecules (antinges displaced tgt with MHC - MHC I - Transmembrane glycoproteins found in almost all nucleated cells - Recognised by cytotoxic T cells - MHC II - Transmembrane glycoproteins found in only the antigen presenting cells - Recognised by T helper cells Exogenous and endogenous Exogenous antigens - Utilized by APCs - Antigens taken up by endocytosis/phagocytosis - Mature into late endosome and fuse with vesicles containing MHC class II proteins - MHC II complex transported to membrane cell surface Endogenous - Utilized by most cells - Proteins produced within cytosol (viral proteins) - Degraded into peptide fragments in cytoplasm - Bale to bind to MHC class I at ER - MHC class I complex is transported to cell membrane Activation and clonal formation T cell activation - Binding of antigen to TCR (T-cell receptors) - Cytokines from Th activates Tc and B cells Formation of clonal populations - Effector cells - Memory cells (longer lifetime up to years) Undesired immune response - Immunocompatibility - device failure due to the accumulation of inflammatory cells - Hypersensitivity - Allergic reaction is defined as unusual,  excessive or uncontrolled immune reaction by acquired immune system Four main groups of immune responses that are undesired Type I : IgE mediated - Caused by plasma cells - Starts immediately within seconds and is resolved within 2 hours - Typical allergic response to environmental factors (pollen) - Rare case for biomaterials - Previous exposure to the antigen results in production of a class of antibodies (IgE) fromm B cell - The igE antibodies bind to the Fc receptors in the mast cells/basophils - Upon reexposure to the antigen, the antigen binds to adjacent iGE molecules, which will bring the receptors (Fc) together - This triggers a signaling cascade and induces release of histamine - What do they cause - Type II: antibody - Antibodies dependent mediated - Destroys cells presenting a foreign antigen - Typical reaction for mismatched blood type during transfusion - IgG/IgM antibodies bind to antigen on the surface of a particular cell type - The antibodies will cause cell destruction - Cytotoxic - What does it cause - Type III immune - Days/weeks after original exposure to antigen complex mediated - Both soluble antigen and antibody must be present - Activate by immune complex (antigen-antibody) formation and then precipitation - Recruitment of phagocytic cells - May be a concern for slow-degrading biomaterials  - Antibodies bind to free flowing antigen (unbounded antigen) to form antibody-antigen complexes - Complement system activated - What does it cause - Type IV: T cell mediated - Also called delayed-type hypersensitivity - Starts about 24-72 h after the 2nd antigen exposure - Action of specialized cells TDTH (Delayed-Type Hypersensitivity T-cell); no antibodies involved - How it works - Upon the 1st exposure, TDTH cells form - The 2nd exposure activates the TDTH cells, which release cytokines to attract macrophages - Macrophages release of lytic enzymes leading to local cell damage - Pre-sensitised T cells Cases of biomaterial induced cellular immune response - The denaturation of native proteins through binding to biomaterial surfaces may result in an undesired immune response since they can be recognized as foreign ones. - Ni, Co and Cr constituents in implants: - Metal ions (haptens) bind to protein/cells  - Macrophage/APC are able to present them as MHC Class II molecules - Triggers T-cell activation (Type IV) - Metal hypersensitivity has been reported to cause failure of certain implants In vitro assays (T and B cells) - Cell adhesion and spreading (1) - Cell death (4) - Cytokine release (2) - Cell surface marker expression ( - Cell proliferation (3) - Lymphocyte transformation tests (LTTs) - Lymphocyte migration inhibition assay - ELISA and flow cytometry Lymphocyte transformation test - evaluates the ability of lymphocytes to proliferate (or "transform") when exposed to a particular stimulus - Test for activation of T cells  - Secretion of IL-2 - Proliferation of T-cells - SI (stimulation indices) - - Method of assesment - Immunofluorescence: find specific surface markers - ELISAs: production of cytokines (IL-2/IL-5) - Proliferation Leukocyte migration inhibition assay - It is used to test cell-mediated hypersensitivity (type IV) reactions to biomaterials. - The method used to assess migration can be Boyden chamber assay - Basis of technique - In culture, cells are in random motion, attracted to chemoattractants. - In the presence of antigens that are specific to them, they migrate slower due to the lost in ability to recognize chemoattractant (migration inhibited) Topics for question 9: Q: What are examples of APC? A: macorphae and B cells Q: Which antibody below is most likely to cause agglutination? A: IgM Q: which cells below are both antigen-presenting cells expressing MHC class II? A: Macrophages and B cells Q: You are evaluating three materials (polypropylene, polyethyleneterephthlate, and polytetrafluoroethylene) for potential use as a vascular graft. How would you determine which material induces the least activation of granulocytes? A: We can test for granulocytes: - Neutrophil - Eosinophil  - Basophil Can use flow cytometry 1) In vitro assay which cultures the biomaterials with equal amounts of granulocytes a) The fluorescent tagged antibodies are used to mark the receptors on the granulocyte surface that indicate activation i) The receptors are basically the MHC class proteins 2) Then use fluorescence activated cell sorting (FACS) to count theamount of cells for each material Q: In experiments to evaluate the tissue response to a biomaterial, a control group is often included involving the complete surgical procedure but without implanting the biomaterial. What is the rational for the inclusion of this control group, and what is the expected tissue response? A: So basically this control group does not have any biomaterials. So a surgical process is done without involving any biomaterials (just implants) to see if there’s any inflammation after the surgery that is caused solely due to the implant (and not the biomaterial) The control group serves as a baseline for the body’s reaction to the implantation process (since injury occurs during implantation, which will activate the innate immunity). The researcher can then compare the level of response to the control (such as acute inflammation and macrophage migration) to the levels from the biomaterial test group. Q: A degradable scaffold material for bone tissue engineering is implanted into the body. Describe how the implantation could result in both exogenous and endogenous antigen presentation by the surrounding cells. What type of T cells are activated in each of these scenarios Exogenous: The degradable components from the material serves as the exogenous antigens. The surrounding APCs (macrophages or B cells) phagocytose the degradable components, and then present them with MHC Class II molecules. MHC class II are recognized by T helper cells. Endogenous: The material can be toxic or carcinogenic, and thus alter the surrounding cells.The diseased cells have endogenous antigens which can be presented with MHC Class I molecules on their cell membrane. MHC Class I is recognized by T cytotoxic cells Q: A poly(lactic acid-co-glycolic acid) copolymer was manufactured into microparticles of 1 and 200 μm in diameter. Discuss the possible response of macrophage cells to these two microparticle samples when they are incubated with the cells A: size of microparticles determines the degradation and responses of macrophages. When the size of microparticles is larger than 5 μm, macrophages cannot phagocytose them and encounter “frustrated phagocytosis.” - Macrophages are unable to engulf the PLGA particles with the size of 200 μm, and they will encounter frustrated phagocytosis. - Macrophages can phagocytose the PLGA particles with the size of 1 μm and digest them Topic 10 Why blood-material interactions - There is a widespread use of blood-contacting devices, such as catheters, dialysis membranes, heart lung machines, vascular prostheses, etc. - Blood-material interactions are the leading cause of device failure. - Hence, there is a need to evaluate the hemocompatibility of biomaterials. Blood circulation in blood vessels Hemostasis - is a process that arrests bleeding. - Basic steps - Vascular constriction at the area of insult - Formation fo platelet plug through platelet activation - Blood coagulation (thrombosis) Platelet (thrombocyte) characteristics - Non-nucleated fragments of megakaryocytes - Small (3-4 microm) and irregular shape - Half Life of 8-12 days - Cannot proliferate (cos no nucleus) - Contains - Mitochondria - Golgi apparatus - Intracellular granules - Store chem mediators for activation - Functions - Formation of platelet plug to reduce bleeding - Stabilizing plug via activating blood coagulation cascade Platelet activaiton 1) Exposure to soluble factors a) Activation by von Willebrand factor (vWF) produced by endothelial cells 2) Interaction with ECM and cells of injured vessel walls a) Platelets adhere to collagen (ECM) What will happen after 1) Shape change being activated? a) Swell b) Irregularly shaped (star like) c) Pseudopodia-like 2) Secretion of bioactive factors a) Granule releases ADP, prothrombin 3) Adhesion and aggregation a) Adheres to injured area b) Interactions of various ligands c) (collagen, vWF, fibrinogen, glycoprotein or integrin receptors) 4) Formation of platelet plug a) Increase platelet aggregation b) Platelet membranes form a catalytic environment (phospholipids) for conversion of prothrombin to thrombin (coagulation cascade) Coagulation cascade - Tissue factor pathway - Is extrinsic, initiated by the release of tissue factor, synthesisedby macrophages and endothelial cells - Contact activation pathway - The contact activation (intrinsic) pathway is initiated by exposure of blood to ECM molecules Common pathway of - Factor V attach to the platelet which acts like a receptor for the coagulation factor Xa to which will convert prothrombin to thrombin cascade - Thrombin cleaves fibrinogen to fibrin, which then polymerise to form basis of bloodclot - Factor XIIIa crosslinks the thrombin firbes which provide mechanical integrity to the clot - Countdown on IN - 12,11,9,8 - There is X in EXTRINSIC - III and VII equals to X  Coagulation control 1) Physiological Factors a) Blood flow can remove activated components or act to dilute them b) Need for membrane surfaces in surface-catalyzed reactions, limiting the initiation of coagulation to traumatized regions 2) Soluble and insoluble biochem factors a) Heparin/anti-thrombin III complex i) Inhibits thrombin by forming a tight complex ii) Prevents exposure of thrombin’s catalytic sites b) Thombomodulin (insoluble) i) found in endothelial surface of blood vessel walls sequesters thrombin to prevent cleavage of fibrinogen to fibrin Questions: - Which molecule can trigger the contact activation pathway in the coagulation cascade?\ - Ans: ECM moleucels (Elastin) Fibrinolysis : thrombus dissociation - It is a process where a fibrin clot is broken down (clot dissolution) to restore normal blood flow to the region. - Endothelial cells produce tissue plasminogen activator (tPA) which converts plasminogen to plasmin - Plasmin cleaves fibrin to dissolve thrombus - The process is balanced by the presence of α2 -plasmin inhibitor which prevents the plasmin from acting on the fibrin - Role of endothelium - Interior wall (thin lining of endothelial cells) - Line blood vessels - Runs throughout our circulatory system - Anticoagulative properties - Heparan sulfate aids thrombin inhibition - Secretes soluble chemical mediators prevents platelet aggregation - Heterogeneous surface - Contains GAGs molecules (e.g. Heparan Sulfate) which form glycocalyx - Contains integrins and receptors that can bind to platelets - Exposure of ECM after injury initiates coagulation [Contact] Test for hemocompatibility - 5 areas of evaluation - Thrombosis - Coagulation - Platelets - Hematology - Immunology - Local and systemic effects - Localized blood clotting has negative impact on the impact on the performance of the device - Systemic effects, e.g., thrombus embolization (pieces of the clot break off and are carried elsewhere in the circulatory system) can result in serious complications such as stroke. In vitro assessment - The period of interaction time is key and it ends until the coagulation occurs. - It can be static (direct exposure of material to blood) or dynamic (closed-loop flow systems) - Quantifiable parameters include - Coagulation time  - Amount of adhered platelet - Mass of the thrombus - Amount of granule released - However needa use anticoagulants which will affect the recorded coagulation time - Design criteria - Inclusion proper control materials - Type of animal - Length of testing and point of time - Assessment methods - Histology to determine thrombus formation - Biochem assays for plasma coagulation factors - Disadvantage - Difficulty in controlling/measuring blood flow conditions in animal models Ex-vivo sunt testing - Material is placed in shunt connected to arteries of animal - Blood flow can be controlled and measured - Native (non-anticoagulated) blood may be used - Long-term response may be examined - BUT model does not replicate an implantation procedure thus response to trauma from surgery cannot be evaluated Infection - the detrimental colonization of a host organism by a foreign species. - Damaged underlying ECM - Bacterial colonization of tissue \ - Resistance of bacteria towards the host defense mechanisms - Transfomation of bacterial species from innoculous to virulent - Persistence of infection - Absense of integratio of biomaterials - Presence of cell damage Common pathogen - Gram positive - Staphylococcus aureus (S. aureus) - Staphylococcus epidermidis - Gram negative - Fungi - Enterobacteriaceae - Pseudomonas aeruginosa Gram-positive bacteria Gram-negative bacteria - Single bilayer phospholipid - 2 phospholipid membrane: cell - The membrane made of membrane and outer membrane with peptidoglycan lipopolysaccharide - Macromolecule on cell can mediate - Thin cell wall made of peptidoglycan binding to ECM - Crystal violet - The thick peptidoglycan layer in cell wall retains the stain Types of infection Superficial immediate infection - Growth of microorganisms on the skin - Immediate after implantation - Usually from skin-dwelling bacteria Deep immediate infection - Presence of infection at implantation site - From proliferating skin-dwelling bacteria Late infection - Months to years after surgery - Due to seeding blood-borne bacteria/pathogens originating from another site Questions: Staphylococcus aureus bacteria are less likely to be involved in - Superficial Immediate Infection - Deep Immediate Infection - Late Infection (cos its not a blood borne bacteria) The stages of infection Attachment - Reversible and non specific (formed through an der walls forces and hydrophobic interactions) - Dependent of surface characteristics of bacteria and substrate - 0 hours Adhesion - Irreversible - specific/nonspecific recceptor/ligand interaction - Strength of interaction is teim dependent - 3 hours Aggregation - Chem interaction - Biofilm - Exudation of an extracellular polysaccharide slime (biofilm)  - Biofilm protects microorganisms from phagocytosis - Provides favorable environment for bacterial growth - 24 hours Dispersion - Shear Forces - As early as 2 days after initial bacteria attachment - Cause spreading of infection locally or secondary infection - 48 hours Cell capsule and biofilm - Outer layer of polysaccharide - Most capsules are water-soluble - Protects bacteria from phagocytosis - Allows bacteria to adhere to surfaces and other cells Biofilm - Exudation of slime - Forms a physical barrier to phagocytosis - Inhibits T and B cell formation, antibody production and bacterial opsonization - Can impart antibiotic resistance - How is it formed? - bacterial colonies are no longer attached to the surface of the material but contained within the polysaccharide slime.  - Physical barrier to phagocytic cells - Inhabit both innate and acquired immune response - Impart antibiotic resistanc Question:. Which Stage of bacterial infection involves specific receptor and ligand binding? Answer: Adhesion Quesiton:. Describe the reason why relatively innocuous bacterial species can be transformed into virulent organisms after biomaterial implantation. Answer: Several steps are required for this transformation, aided by the existence of biomaterial/damaged underlying ECM as a surface for attachment of bacteria. The adherent bacteria become resistant to host defense mechanisms and antibiotics. [Superficial Immediate Infection] The biofilm can form as the physical and chemical barrier to the innate and acquired immune system and to the actions of antibiotics. In addition, the constant presence of the biomaterial may exhaust the granulocytes’ ability to kill or phagocytose the pathogen. [Biofilm]. Why? Cos the biomaterial is recognised as a foreign entity, which activates immune system. Granulocytes (especially neutrophils) are continuously recruited to the site to attempt to "neutralize" the biomaterial. However, biomaterials are often too large or chemically resistant to be engulfed or degraded by granulocytes. Therefore will undergo frustrated phagocytosis. Constant activation and unproductive attempts to deal with the biomaterial can exhaust their ability to respond effectively. Biomaterial design Things to consider: - Bacteria resistance - Surface which will repel bacteria - Biomat surface properties - Affect cell morphology, adhesion or motility - What to consider: - Surface hydrophobicity - MATH - Microbial adhesion to hydrocarbon - - HIC Contact angle measurement - Surface charge - Electrophoretic mobility test - EIC - Steric hindrance - Surface roughness - Characterization techniques  - Contact angle - SEM - Cell friendly - Modify surfaces to improve cell adhesion - Media - Proteins in media affect nature of biomaterial surface coating Surface hydrophobicity tests Microbial adhesion 1) Test tube filled with to hydrocarbon a) Water (MATH) b) Hydrophobic hydrocarbon based solvent 2) Bacteria is then added into the test tube 3) The migration of bacteria from water to solvent is recorded by measuring the turbidity of water over time 4) Can tell hydrophobic nature of bacteria overtime Hydrophobic 1) Affinity chromatography interaction 2) Bacteria suspended in aqueous phase andpassed through chromatography column containing hydrophobic material 3) Measure the remaining bacteria int he aqueous phase exiting the column 4) More quantitative than MATH - The hydrophobic packing materials has ligands attached on its surface - Surface charge test Electrophoretic mobility test 1) Microbes are introduced to gel electrophoresis using buffer solution of a known ionic charge 2) Electric field applied and velocity of the microbes migrating is kown 3) Velocity = surface charge Electrostatic interaction 1) Liquid chromatography chromatography 2) Ion exchange chromatography 3) Column is catioionic or anionic packing material 4) Degree and nature of charge of the microbe can be determined In vitro models (bacteria adhesion) - Quantification under static and well-defined flow conditions  - Via microscopic visualization - Manual counting/image analysis software - Use of both small and large animals Ex vivo - Use of shunts Other assessments - SEM and histology - Additional hemocompatibility tests can be included - Bacterial cell wall antibodies produced/found in the blood can be quantified via ELISA over time Question: Answer: - According to the MATH assay, bacteria is likely to be hydrophobic - Therefore, plasma treatment will make it hydrophilic by introducing carboxyl and amide groups - Therefore adhesion of bacterial strain will decrease after plasma treatment of the surface of the biomaterial Q:. Which molecule triggers the contact activation pathway in the coagulation cascade? A: Elastin (ECM) Q: Staphylococcus aureus bacteria are less likely to be involved in A: Late infection Q: Which Stage of bacterial infection involves specific receptor and ligand binding? A: Adhesion Q: Describe the reason why relatively innocuous bacterial species can be transformed into virulent organisms after biomaterial implantation A: One of the ways of transformation requires the adherent bacteria to form non-specific or specific ligands with the surface of the ECM that is damaged. THis is moved on following with the formation of biofilm over the bacteria which acts as a protective layer against phagocytosis and antibodies (action of B cells and T cells). The presence of biomaterial will also cause granulocytes to lose its ability to act on the pathogen (exhaustion)