Introduction to Biomaterials A biomaterial is a nonviable material intended to interface with a biological or physiological system in order to evaluate, treat, augment, or replace any tissue, organ, or biological or physiological function Examples: pacemaker, prosthesis Donated organs are NOT biomaterials Example: eyeglasses vs. contact lens Biomaterials science and engineering is the study of the relationship and integrated roles between processing parameters, structure and composition, and properties on the performance of a material and a biological or physiological system of a particular application. Example: parameter pyramid Processing is the steps for handling and converting materials (and biological components) to form the desired shape of the biomaterial Examples: patterning patch of adhesive protein on cell culture plates Criteria: how do the processing conditions (temperature, pH) affect the components? Structure is the spatial arrangement of atoms within a material or biological or physiological system Examples: FCC or BCC, helix or sheets or random coil for proteins Composition is the chemical makeup of a material or biological or physiological system Examples: atoms in biomaterial (titanium, gold), molecules in the biological component (amino acids) Properties are the behavior or response of a material and/or biological or physiological system Examples: mechanical properties (yield point of a material), binding or adhesion strength of particular proteins for particular cells Criteria: environment (temperature, pH, applied forces) For bio-related applications of materials, one must consider how the viable components of the biological or physiological environment respond to the presence of a foreign, nonviable (synthetic) material For medical applications (implanted, injected), one must be concerned with in vivo (in the living) responses Example: inflammatory responseimmunological responsereject of biomaterial could occur or infection could occur For biotechnology applications (cell culture dishes), one must be concerned with in vitro (outside the living body in an artificial environment) responses to the material Important broad considerations for biomaterials:1. Cytotoxicity involves the release of chemicals from a material that kills cells directly or indirectly2. Biocompatibility is the ability or capacity of a material to perform with an appropriate host response in a particular application3. Healing (for implanted biomaterials) invokes a short term inflammatory response as particular leukocytes (WBCs) are recruited to the injury site4. Ethics minimize the risk to patients and considerations for animal testing; thus, the development of biomaterials involves the following testing stages:a. In vitrob. In vivo tests in healthy animalsc. In vivo tests in model animals of particular disease or impaired stated. Clinical trials (various stages)5. Regulation is the time and cost considerations for implementing novel biomaterials which meet test standards set forth by the following agencies:a. FDA (Food and Drug Administration) provides approval for a specific device, not for a general biomateriali. Example: contact lens vs. glassesb. ASTM (American Society for Testing of Materials)c. ISO (International Organization of Standardization) Traditional approach (before 1970s) was biomaterials were designed to be inert with respect to the physiological surroundings Modern approach is to rationally design biomaterial to interface in a favorable, even dynamic, way with the hostClassification of Materials HandoutOrganelles HandoutNucleic Acids Handout DNA (helix) depends on histones to pack efficiently (complementary bases); whereas, RNA (single strand) have self-complementary strandsAfter Binding to Biomaterial What happens to cells after binding or attaching to the biomaterial via nonspecific or specific forces?1. May affect general housekeeping activities of a cell2. May affect cell viability (ability to live)a. As cytotoxicity of biomaterial increases, cell viability decreasesb. If viability of a cell is compromised to a great extent, the cell may diec. 2 routes to cell death:i. Apoptosis: programmed cell death driven by the nucleus of the cell as part of the bodys attempt to maintain homeostasis (normal status of the host); typically involves the binding of specific ligands that signal a cell to die1. The preferred route because there are no consequences with respect to inflammatory responseii. Necrosis: cell death marked by significant changes to the cell structure and cell membrane permeability1. Cell often undergoes cell lysis (cell ruptures and releases its contents)3. May affect cell stabilitya. Labile cells can replicate or proliferate on a regular basisb. Permanent cells are highly specialized cells that do not readily undergo replication (mitosis)i. Examples: neurons, heart cellsc. Stable cells are somewhat specialized, but can be cued to undergo mitosis under particular circumstances4. May affect cell specializationa. These specialized cells may be part of a particular tissue, organ, etc.b. Cellular differentiation is the series of changes in a cell (normally involving gene expression) by which a cell becomes specializedi. Progenitor cells are nondifferentiated cells that are able to renewii. Totipotent stem cells can differentiate into any type of cell or cell phenotypeiii. Pluripotent stem cells can differentiate into many, but not all, cell phenotypesiv. Embryonic stem cells are usually totipotentv. Adult stem cells are usually pluripotent1. Hematopoietic stem cells are derived from the bone marrow; form blood cells2. Mesenchymal stem cells originate and differentiate into cells as part of connective tissue (tendons, ligaments)c. Stem cellscellular differentiationcommitted cells (specialized)d. All cells in a host have the same genetic code (genome), but as cells differentiate, some subset of genes are accessed in order to allow some subset of genes to be preferentially expressed, while others are turned offi. Results in distinctive behavior (biological profile) of a cell to give rise a particular cell phenotypeii. A cell phenotype can be determined by features such as the cell shape (cell morphology)iii. However, cell phenotypes can always be determined by its gene expression profile5. Adherent cells (cells that differentiate and attach to a biomaterial) may organize themselves into tissuea. Requires a source of nutrients and waste disposal, particularly if multiple layers of cells are involvedb. Angiogenesis is the formation of small blood vessels6. Biomaterial in contact with cells, or even just near cells, may cause a distinctive reaction by the host to this foreign material or invader which may involvea. An inflammatory response which is an attempt by the host to dispose of foreign invadersb. Fibrous capsule formation: cells of the host attempt to isolate the foreign invader by forming a protective barrier-ultimate resolutionSpecific vs. Nonspecific Forces or Interactions (Energy) Nonspecific Forces Ubiquitous and does not involve recognition between particular functionalities or chemical groups Origins: primary bonds (covalent, ionic/coulombic or electrostatic, metallic), secondary bonds (hydrogen, van der waals), and other bonds (hydrophobic, hydrophilic, steric) Nature: Can be attractive or repulsive Can be large or small in magnitude Can be long-range or short-range in terms of distance dependence DLVO Model: Involves van der waals and electrostatic forces Accounts for geometry size and composition of macrobodies but not the steric interactions Biomaterials are typically subject to nonspecific attractions to proteins : protein adsorption Can involve undesirable subsequent interactions with leukocytesinflammationbiomaterial rejection To overcome nonspecific attractive interactions between protein and biomaterial, need to promote nonspecific repulsive interactions between protein and protected biomaterial by adding source of steric repulsion (PEO, PEG) Specific Forces Particular to a system (biological or physiological) Involves recognition between 2 or more particular adhesive groups In case of cells, these matching adhesive groups or functionalities can be referred to as a receptor-ligand pair: typically macromolecules (proteins, glycoproteins) Ligand: often soluble species, but can also be mobilized on a second cell Receptor: often present on cell membrane typically immobilized, but can undergo lateral diffusion Examples: folate receptor-folic acid, antibody-antigen Origins: lock and key analogy1. Particular structure to form the binding pocket in receptor for matching ligand2. Favorable orientation between binding pocket of receptor and matching domain of ligand 3. Close association or contact between receptor-ligand pair4. Involves nonspecific forces, but localized to particular regions of binding pocket and domain of ligand (induced fit) Nature: Usually attractive Can be large or small in magnitude Only short-range Receptor-ligand model accounts for binding and unbinding events between soluble pairs, but not collective receptor-ligand interactions between 2 cells High kA favors binding and low kA represents weak affinity between the receptor and the ligand 3 routes for an implanted biomaterial Unmodified surface: nonspecific attractive interactions with proteins are likely Surface modification #1: add grafted PEO chains to surface Surface modification #2: to promote specific interactions with particular proteins or even cells (phenotypes) by functionalizing biomaterials with particular ligandsExperimental Assays to Measure Adhesion Strength, Cytotoxicity, and Viability Common methods to measure the adhesion strength between cell and biomaterial involve measuring the critical shear stress necessary to detach 50% of adhering cells from a biomaterial (plot) Methods to measure adhesion strength:1. Spinning disk: cells at edge are most likely to fall off2. Parallel plates in a flow chamber: cells are either on top of bottom plate or on bottom of top platea. Cells are exposed to a small, but non-zero, fluid velocity so that only weakly adherent cells with detach In vitro tests Migration assays track the time dependent movement of cells Individual cells (cells might move in random pattern) Translocation speed is the speed of a cell in one general direction before it changes direction Persistence time is the time that cell spends moving in one general direction before drastically changing direction The translocation speed and persistence time can be affected by adding soluble factors in one area of the sample chamber (adding chemical gradient) Soluble factor will eventually diffuse within fluid Adding chemical gradient promotes chemotaxis: preferential path due to soluble factors Groups of cells Capillary tube test (Figure 9.41) Boyden chamber assay (Figure 9.43) Cell morphology assays If A has smaller spread area than B, B cells are more viable Adhesion of cells is lower in A than B Nonspecific: if plasma membrane is a negatively coated surface with positive charge, make plasma hydrophobic Specific: add ligand Cytotoxicity assays are important in determining the overall biocompatibility of biomaterial Often arise from chemical factors from biomaterial (ions, molecules that have leached out of biomaterial; leftover monomer or other reactants from biomaterial; corrosion by-products) Other factors include surface topography, biomaterial size and shape Many cytotoxicity assays are qualitative in nature (monitor changes in cell shape), but many are quantitative in terms of measuring cell viability For quantitative assays Use a dye that selectively labels only the dead cells OR use vital stain to only label live cells Necrosis-affected cells are more likely to allow this Measure intracellular enzyme that is now present in extracellular space due to cell death Necrosis more likely to allow this as well Example: lactate dehydrogenase (LDH) 3 types of in vitro cytotoxicity assays for measuring cell viability1. Direct contact assay places the biomaterial in direct contact with the cells and divide into separate onesa. Sample chamberplace cells on chamber wallplace biomaterial on top of cellsb. As time passes, see how cells have migrated by splitting plate into circular zones to measure spread area of cellsc. Placing biomaterial may crush cells (many cells in middle zone usually die from trauma instead of cytotoxicity effects)2. Agar diffusion assaya. Agar is soft hydrogel that allows permeations and acts as a physical barrier and diffusion barrierb. Cells are no longer subject to the trauma from direct contact, but are exposed to soluble factors escaping from biomaterial3. Elution or extract assaysa. Put biomaterial in solventb. After certain time interval when products from biomaterial has leached into solvent, remove the biomaterialc. Pour supernatant into a chamber with cells presentIntroduction to Proteins Proteins are polymerized amino acids (amino acid~monomer unitcondensation polymerization (lose water)polymer chains (peptide, polypeptide, or protein) Peptide: short chain of amino acid residues (typically too short to possess a 3D structure) Polypeptide: longer chain of amino acid residues (typically has a defined length and sequence) Proteins: usually refers to natural polypeptides of significant length and weight (have a defined 3D structure under physiological conditions that helps promote its particular function) Amino acids: building blocks for proteins (usually find 20 different types of amino acids in nature) Structure: N-terminus is the basic group (accepts hydrogen ions), and C-terminus is the acidic group (donates hydrogen ions) Amphoteric: possess both cationic and anionic charges R groups can be 1 of 20 possibilities that fall into 1 of 4 types of amino acids Nonpolar (R=CH3, XCH3) Polar (R=XOH, XSH, XCONH2) Acidic (R=XCOOH or XCOO-) Basic (R=XNH2 or XNH3+) Polymerization of amino acids occurs via condensation reaction to form a peptide bond With 20 amino acids, many combinations of sequence composition and length depending on solution conditions give rise to protein function Protein structure Primary structure depends on linear order of amino acid residues in a chain For natural proteins, primary structure is dictated by codons in genome Codons are sets of 3 oligonucleotide bases which code for a particular amino acid Recall: protein synthesis steps DNA sequence information is encoded in mRNA via transcription mRNA is used to synthesize a protein via translation Secondary structure arises from interactions between neighboring amino acid residues Random coil has no regularity in structure helix: 3.6 amino acid residues per turn; single-stranded; R groups face the outside of the helix with hydrogen bonding occurring between every 4th R group; hydrogen bonding is not very strong, allowing flexibility Example: myosin proteins in muscles have helices sheets allow for lamellar chain folding; stabilized by hydrogen bonding between folded domains; found in semicrystalline polymers Parallel vs. anti-parallel R groups tend to be small to minimize steric hindrance to sheet formation Hydrogen bonds are very important Extended chains resist further extension Example: fibrin protein which is part of silk Tertiary structure: 3D arrangement of a polypeptide chain due to folding Folding exposes particular amino acid residues to surrounding solve, hides particular amino acid residues, and/or ultimately determines the formation of binding pockets for receptor-ligand to bind to Gives rise to particular functions of proteins How protein folds into particular tertiary structure depends on primary and secondary structures as well as the types of bonds and interactions on neighboring amino acid residues Bond types Primary: ionic bonds between acidic and basic groups and covalent bonds between cysteine groups forming disulfide bonds Secondary: hydrogen bonds and van der waals Other types: hydrophilic (molecules that prefer to be solvated by water molecules) and hydrophobic (molecules that tend to exclude water molecules) Note that folded structures are dynamic and depend on solution conditions as well as its bound states (induced fit that occurs in receptor-ligand binding events) Tertiary structure can be disrupted (denatured) if the surrounding conditions change or if you introduce a biomaterials surface Denaturation of proteins is typically irreversible, but renaturation is possible for oligonucleotides In its natural (native) state, many proteins have the simplest tertiary structure known as globular (essentially spherical in shape) Quaternary structure results from having multiple polypeptide segments (subunits), each with their own primary, secondary, and tertiary structures Example: Hemoglobin is an oxygen-binding protein on RBCs The primary structure of normal vs. sickle cell hemoglobin protein differs by only one amino acid residue out of 149 amino acids (valine instead of glutamic acid) Causes RBC to have a sickle cell shape that interferes with its function Protein absorption is important to biomaterials because it is likely to alter the structure and function of the protein, which could alter the biomaterial Surface that resist, hinder, or at least delay protein adsorption events from occurring are known as nonfouling surfaces (or stealth surfaces) Nonfouling surfaces are often desirable because they prevent the subsequence recruitment of inflammatory cells, but they also prevent the recruitment of desired cell phenotypesThermodynamic Factors for Protein Adsorption Events Adsorption refers to the adherence or binding of a species to a surface Absorption refers to the uptake of a species into the bulk of a material Gads=H-TS Gads represents the change in Gibbs free energy due to a change in state from nonadsorbed state to adsorbed state H represents the change in enthalpy (bond formation greatly affects enthalpy or internal energy of the system) T is the absolute temperature S is the change in the entropy As Gads becomes more negative, the event becomes more favorable Favoring adsorption events H is negative: arises from bond formation between protein and surface (ionic bonds and van der waals) S is positive: water deadsorption or dehydration Opposing, preventing, or hindering adsorption events H is positive: resistance to release bound water molecules (hydrophilic surface) S is negative: steric repulsion at the surface due to adsorbed or grafted polymer chains Common synthetic polymer that is used in nonfouling surfaces is PEG Though nonfouling surfaces are often desirable, immobilization of a functional protein to a surface is often necessary for many biomaterials applications or biosensors and characterization techniques (ELISA) One approach to immobilize a protein in a controlled manner involves using amino acid residues not directly involved in a particular function Native protein with hydrophilic and hydrophobic domains as well as a binding pocket for specific ligandallow for uncontrolled protein adsorption to biomaterial surfacedenatured protein does not preserve desired function (hydrophobic domains bind to hydrophobic biomaterial surface) Protein with cysteine groupscontrolled immobilizationthiol groups on cysteine residues have high affinity for gold surfaceKinetics of Proteins Key considerations1. Transport mechanism for protein to arrive at surfacea. Diffusion is the random movement of atoms, molecules, macromolecules, etc. through its surroundings (fluid, tissue)i. Macrobody diffusion described using Stokes-Einstein equation ii. D=diffusion coefficient (diffusivity), k=Boltzmanns constant, T=absolute temperature, r=radius of macrobody, =viscosity of fluidiii. Can describe Brownian motionb. Thermal convection: transfer of potential energy (heat) via currents within a fluid phasec. Flow is the continual movement of a fluid medium under stress (blood flow through blood vessles)i. Diffusion and flow are the primary transport mechanismsd. Coupled transport typically involves 2 individual transport mechanisms (diffusion+flow)i. ii. v=velocity, Q=volumetric flow rate, b=distance between plates, w=width of plates, y=distance of reference object from 1 walliii. relies on flow initially, then relies on diffusion to get to the wall2. Mass or concentration of protein3. Affinity (specific or nonspecific) of protein for surface4. Presence of other competitive protein speciesa. For 2-4 collectively, if protein A has low affinity for surface and is present in solution at high concentration, and if protein B has high affinity for surface and is present in solution at low concentrationi. Protein A will begin to occupy available sites (adsorption)ii. Adsorption rate will decrease as fewer sites are availableiii. Surface is saturated with protein Aiv. Competitive displacement of protein A by protein Bb. Know the plotsc. Replacement of weaker adsorbate by a strong adsorbate species over time is called the Vroman effect5. Conformational and biological changes in adsorbate (species that has adsorbed to surface)a. Change in protein structure often involves denaturation of proteinb. Nonspecific affinity of protein for biomaterial tends to increase asi. Size of protein increasesii. Net opposite charge between protein and surface increasesiii. Lack of net charge on protein and/or biomaterialiv. Protein structure becomes more static upon adsorption to surfaceSeparation and Identification of Proteins in a Mixture Basis of protein separation from a mixture Size (and/or molecular weight) Affinity for a substrate Charge Separation based on size alone Size exclusion chromatography (SEC) Proteins migrate through a column of porous resin beads using gravity (and/or pressure) as the force Larger species take less time (only needs to go between beads) Smaller species take more time (have to go through and between the beads) Solvent is the mobile phase and beads is the stationary phase Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) SDS is a surfactant oligomer used to make intrinsic net charge to provide an equivalent charge:mass ratio for all proteins Protein denatures into more linear conformation Each SDS oligomer binds to 2 amino acid residues in a regular fashion Proteins migrate through a continuous hydrogel (highly entangled, cross-linked polyacrylamide chains) Smaller ones elute faster Separation based on affinity Specific binding to immobilized ligand on resin bead Nonspecific affinity based on hydrophobic attractions between hydrophobic bead and hydrophobic domains of protein of interest Separation based on charge Ion-exchange chromatography Identification of protein of interest Chromatography is based on color changes due to photon absorption characteristics of macromolecules, often at a particular wavelength of light Employs chromophores which are sensitive (chemically) to light Colorless chromophore prior to light exposureadd enzyme to drive a light-sensitive reactioncolor change due to photon absorption Fluorescence is based on photon emission characteristic of macromolecules or dye molecules attached to macromolecule of interest Fluorophore is usually molecule dye that reaches an excited electronic state through an excitation event, then emits photon upon relaxation to lower energy state or ground state Western blotting Separate via SDS-PAGE Transfer the bands of separated protein species onto nitrocellulose paper Fluorescence tagging of protein of interest Wash off any unbound ligand ELISA (Enzyme-Linked Immunosorbent Assay) Separate via SDS-PAGE or affinity chromatography Plate well for introducing a solution of protein Use enzyme-linked chromophore on ligand to tag the protein of interest Wash off any unbound ligand Add light to catalyze reaction Sandwich ELISA Add ligand #1 to the wells Add separated protein and wash off any unbound protein Add ligand #2 that has enzyme-linked chromophore Wash to remove any unbound ligand #2 Add light to catalyze reactionSeparation and Identification of Oligonucleotides in a Mixture Southern blotting: often used to identify sequence coding for a particular gene Denature DNA duplexes and add base (NaOH) Electrophoretic separation (PAGE) No SDS is needed because already equal mass:charge ratio Identify the sequence of interest using fluorescently-tagged complementary sequence Wash away any unbound sequences Northern blotting: separate and identify RNA sequence Electrophoretic separation Identify using fluorescently-tagged complementary sequence Amplification of oligonucleotides Polypeptide synthesis is complicated, but if you have at least one copy of an oligonucleotide sequence, you can copy or amplify the number of sequence strands using polymerase chain reaction for DNA or reverse-transcription PCR for RNA Steps for PCR DNA duplexdenature via heatadd primers (short complementary sequences) and monomers of A,T,G, and C (nucleotides)cool solution to allow for hybridizationadd polymerase to form phosphodiester bonds between nucleotidesduplicated duplex Amplify the number of copies of a duplex by 2x where x is the number of cyclesOverview of Hosts Defense System The immune system is triggered by injury to defend the host against infections, organisms, and foreign material and to maintain or return to homeostasis Self (host-derived) vs. non-self (foreign) Foreign material could be (virus, bacteria), biomaterial, host cells infected by foreign pathogen, etc. Extent of injury depends on chemical nature, size, shape, surface roughness, and other physical properties of biomaterial Extent of immune response depends on the nature and extent of injuryTypes of Immune Responses Innate/intrinsic immunity Born with it Nonspecific in nature: host identifies a species as non-self (foreign), but does not recognize specificity of the foreign pathogen 1st line of defense Adaptive/acquired immunity Generate a memory for past foreign invaders Specific in nature: body prepares a particular set of cells (lymphocytes) that recognize particular elements of particular foreign pathogens Invoked if the innate response is unsuccessful in clearing the host of a foreign material Other general differences between innate and adaptive immunity Physical barrier Innate: skin, mucosal lining (nose) Adaptive: lymphocytes in epithelia Chemical and other physiological barriers Innate: body temperature (fever), antimicrobial proteins, low pH in stomach Adaptive: antibodies (secreted by B cells) Other chemical barriers Both have cytokines: cellular proteins (usually secreted) which affect the behavior of other cells Examples: interleukins (IL)-cytokines secreted by leukocytes (WBCs) Lymphocytes are a subset of leukocytes Chemokines: chemoattractant proteins that control the adhesion, chemotaxis, and activation of leukocytes/lymphocytes Types of cells involved Innate: leukocytes, neutrophils, eosinophils, basophils Adaptive: lymphocytes (T cells for cellular immunity and B cells for humoral immunity) Cells that can act as intermediaries between innate and adaptive immunity Innate: macrophages internalize pathogens via phagocytosis and digests the pathogens Adaptive: macrophages present digested foreign antigen on plasma membrane to signal specific lymphocytes (aka antigen-presenting cells)Types of Leukocytes Granulocytes or polymorphonuclear (PMN) leukocytes Granules in the cytoplasm identified via staining Irregularly shaped multiple nuclei Main function is to phagocytose foreign material as well as lysed cell parts Example: neutrophils, basophils, and eosinophils Neutrophils die very quickly following their activation and phagocytic activity Macrophages also phagocytose foreign material, but they do not die quickly in case they need to act as APC Monocytes One nucleus Circulate throughout bloodstream and usually only differentiate when recruited to infected, inflamed area or as they enter tissue Often differentiate into macrophages Phagocytic capabilities, but macrophages have more capabilities Lymphocytes (particular to the adaptive immune system) T cells originate in thymusactivationeffector T cells used in cellular immunity B cells originate in bone marrowactivationeffector B cells (plasma cells) secrete antibodies Megakaryocytes Originate in bone marrow If activated, they break up into smaller fragments called platelets along clotting cascade Overview of Inflammation Typically the first defensive response of a host to a biomaterial always involves an inflammatory system Acute inflammation is an immediate response of short duration (hours, days); typically a normal response that persists for days to years General undesirable because no resolution of the injury is reached via chronic inflammation Cardinal signs1. Tumor (swelling) is caused by recruitment of inflammatory cells (neutrophils, macrophages)2. Dolor (pain) is caused by kinins release during injury (blood clotting cascade)3. Calore (heating) is due to increased blood volume; part of the bodys attempt to kill foreign pathogens near injury4. Rubor (redness) is due to increased blood volume near injury site Steps Activated tissue macrophages secret cytokines (IL-1, IL-6, TNF-) which increase vascular expressions of CAMs to recruit neutrophils and other inflammatory cells IL is interleukin and TNF is tumor necrosis factor Nearby blood vessels vasodilate or expand in diameter to allow blood volume to increase and thus, faster recruitment of inflammatory cells to injury site Neutrophils are recruited and activated for phagocytosis (typically occurs within 1st few hours following vasodilation) Activated macrophages are recruited Phagocytosis typically occurs 5-6 hours following injury Conditional: IF the injury is resolved, terminate acute inflammation by decreasing inflammatory stimulus (turn off inflammatory stimulus by introducing competing, but non-inflammatory inducing cytokines such as IL-1ra) ra stands for receptor agonist competes with IL-1 for receptor on inflammatory cellsCAMs Handout Selectins Glycoproteins which recognize specific carbohydrate groups called mucins Some are expressed on inflamed vascular endothelial cells (E-selectin, P-selectin) Some are expressed on inflammatory cells (neutrophils) Responsible for initial, weak adhesion events between leukocytes (neutrophils) and vascular endothelial to drive neutrophil rolling along vasculature near injury Mucins Ligand to selectins Integrins Heterodimeric proteins present on leukocytes (neutrophils) Bind strongly to their ligand known as immunoglobulin (Ig) superfamily Expressed by inflamed endothelial cells near injury ICAM-1, VCAM-1 Equilibrium constant Important cytokines responsible for vascular changes during inflammatory response TNF-, IL-1, IL-6 Secreted by activated macrophages Increase CAMs expression level on vasculature (endothelial cells) Increase vascular permeability Important chemokines responsible for activating inflammatory cells IL-8 Induces a conformational change in integrins to allow binding to its ligand (Ig superfamily)Neutrophil Recruitment1. Rolling of neutrophils along endothelial cells due to transient adhesion events from weak mucin-selectin binding events2. Arrest or stop neutrophil rolling via strong or tight binding events between integrin-Ig superfamily receptor-ligand pairs3. Diapedesis is the movement of neutrophils across vasculature by squeezing between leaky endothelial cell junctionsa. Favored by increased vascular permeabilityb. Favored by chemoattractants (subclass of chemokines) especially IL-8 and MIP-1b which cause chemotaxis4. Transendothelial migration is the process of movement across endothelial cell lining through surrounding tissue to actual injury site Extravasation is the entire series of steps involved in recruiting neutrophils from blood stream to injury site Role of neutrophils Following their arrival at injury site Phagocytosis of foreign material Respiratory burst: release of reactive O2 and N2 series to kill foreign pathogen Role of macrophages Residual tissue macrophages are already present Additional macrophages are recruited following neutrophil recruitment (source is the circulating monocytes that differentiate into macrophages) Actual functions Phagocytosis: macrophages have the highest capacity for phagocytosis of foreign materials Release of cytokines to upregulate key CAMs on endothelial cells near injury that are necessary to recruit other leukocytes Conditional: IF injury is not resolved via acute inflammatory response, macrophages serve as APCs to recruit lymphocytes as part of AIRNormal Wound Healing Acute inflammation and blood clotting cascade (simultaneous) Short duration Primarily involves neutrophils and macrophages Phagocytosis (engulfment) and degradation of foreign material (dependent on size of biomaterial) Frustrated phagocytosis by macrophages involves release of chemically degrading products in an attempt to degrade or breakdown larger biomaterials Formation of granulation tissue (2-5 days following injury) Macrophages and fibroblasts of vascular endothelial cells are recruited to the injury site to form granulation tissue Granulation tissue has pink, soft appearance and is specialized tissue which is a hallmark of inflammation that is healing Role of phenotypes Macrophages secret chemoattractants to recruit fibroblasts and endothelial cells Fibroblasts synthesize extracellular matrix proteins (proteoglycans, collagen) and later forms fibrous capsule Endothelial cells are responsible for angiogenesis by promoting building of new blood vessels from existing ones Foreign body reaction Involves granulation tissue and FBGCs Foreign body giant cells (FBGCs) are fused macrophages and monocytes Can phagocytose larger objects Typically around injury site for the lifetime of the implant Fibrous encapsulation Comprised of mature granulation tissue consisting of larger blood vessels and aligned collagen fibers Marks end of healing stage Serves as barrier between biomaterial and host (physiological environment)Factors Involved in Injury Size of biomaterial As size increases, more frustrated phagocytosis (mostly macrophages) and more FBGCs form for more phagocytic capabilities Shape Surface area More surface area means more FBGC formation Less surface area means less FBGC formation and more granulation tissue formation Implant site Splinter in the skin (no blood, but painful) If injury involves only epithelial cells, then extrusion may occur in which the skin forms a pouch around the material to extract it out If the injury involves labile or stable cells, then either fibrous encapsulation or regeneration is possible in which damaged cells are replaced by cells identical to the native tissue If injury involves permanent cells, repair is possible in which damaged cells are replaced by cells that are different from original native tissue (scar tissue formation is likely) Mechanical factors such as rubbing, shedding, wearing into smaller pieces Chemical nature of biomaterial If material is biodegradable, material may completely disappear and undergo resorption If material is not degradable and recognized as foreign, fibrous encapsulation is likely If material is not degradable, but can completely interface with physiological surrounds, integration may occur without fibrous encapsulation Fibrous encapsulation of implanted biomaterial is the most common resolution (new equilibrium state or homeostasis redefined for host)Repair vs. Regeneration For superficial wound involving the epidermal layer, regeneration may occur Epithelial cells near wound degrade surround extracellular matrix and then flatten out to cover some of the wound Migration and proliferation of epithelial cells to wound are to cover any remaining exposed areas Epithelial cells resume their normal morphology and now have to reattach to extracellular matrix For deep wounds in the dermal layer, repair will occur Acute inflammation and blood clotting cascade which forms a barrier to stop bleeding and prevent foreign pathogens from entering wound area 14 clotting factors and platelets form a polymerized fibrin network (scab) Formation of granulation tissue involving influx of fibroblasts Deposit collage fibers (type III) in random orientation near extracellular matrix Dissolve fibrin clot via enzymatic cleavage events and scab falls off Scar formation due to constant remodeling of extracellular matrix Randomly oriented collage type III fibers replaced by oriented collagen I fibers Collagen accumulation and remodeling occurs for 2-3 months Chemical and physical properties of biomaterial may induce a persistent inflammatory stimuli (chronic inflammation) Undesirable because it doesnt resolve injury Can last weeks, years, to lifetime of host Macrophages, monocytes, lymphocytes, and granulomas (layer of FBGCs) involvedIn Vivo Testing of Inflammatory Response In vivo testing first occurs in live animal models Starts off with rats, mice, rabbits Later tests in sheep, dogs, cows, pigs Important factors include choice of animal, implant site, and size of biomaterial Response is examined at particular time intervals Acute toxicity effects are negative effects within 24 hours following administration/implantation Subacute toxicity effects are within 14-28 days Subchronic toxicity effects are within 90 days Chronic toxicity effects are after 90 days Choice of administration of biomaterial Direct contact: intact biomaterial makers intimate contact with surrounding tissue Extracts: pieces of biomaterial are implanted Cage implant: biomaterial is housed in a stainless steel cage to examine the inflammatory response in the absence of contact between biomaterial and surrounding tissue Assess in vivo inflammatory response via histology (taking tissue sections to examine cell phenotypes present, tissue present, etc.)Hallmarks of Acquired/Adaptive Immune Response Involves specific recognition of particular foreign antigen (usually involve peptide fragments from foreign pathogen) Each nave (not yet exposed but has capabilities) T cell and each nave B cell can recognize a specific foreign antigen or unique antigen In contrast, innate immunity only recognizes general molecular patterns of foreign pathogen Diversity in lymphocyte populations Due to genetic rearrangement during development and maturation of nave lymphocytes Large and diverse lymphocyte population that results If AIR is needed, APCs must find the particular T and/or B cell that recognizes the antigen present Distinction between self and non-self: nave T and B cells that recognize self-antigens are quickly destroyed during a selection process Immunological memory Nave T/B cellsactivationeffector T/B cells and memory T/B cellsmitosisclonal populationsuccessful cellular/humor IRregulation process to eliminate many effector cells, but leave memory cells alone in case same pathogen invades againmemory cells outlive effector cells Antigen: antibody generator (Ag) Antibody: immunoglobulin type of protein (Ab) Secreted by plasma cells and immobilized on B cells Ab binds to a particular binding domain of a Ag known as an epitope Types of AIR Humoral immune response Plasma cells Soluble antibodies secreted by plasma cells Basic structure of typical bivalent (2 binding sites): heavy chains, light chains, disulfide bonds, hinge region, antigen-binding fragment, constant fragment Antigen-binding fragment binds to antigen to prevent antigen from its intended activity Constant fragment binds to particular receptors (FCR) on macrophages to alow phagocytosis of Ag-Ab complexes Role of secreted antibodies Antibody binding to antigen to inactivate it and prevent it from infecting a host cell Antibody-antigen complex is marked for phagocytosis by macrophages Antibody binding to antigen can invoke complement system: set of proteins can self-assemble on pathogen surface to puncture a hole Cellular immune response T cells can split into helper T cells and cytotoxic T cells Cytotoxic T cells (CD8 receptor) recognize and kill non-self or otherwise altered host cells Helper T cells can either help to mediate immune response of plasma cells or help cytotoxic cells (CD4 receptor)Survey of Homeostasis Homeostasis: normal physiological activity in the absence of injury to retain the healthy status of the host Hemostasis: mechanisms by host to arrest or stop bleeding which involves the formation and ultimate dissolution or degradation of blood clot Thrombosis refers to the formation of a blood clot (thrombus) In the absence of biomaterials, thrombus can form in blood vessels In presence of biomaterials, thrombus can form on the biomaterial itself Local/systemic effects of thrombosis according to where it forms Hemostasis overview Injury occurs: damage to blood vessels and surrounding tissue Platelets migrate to injury site (activated by binding to exposed collagen) Activated platelets release granular contents containing chemotactic factors that activate and recruit other platelets to injury site as well as other molecules (fibrinogen monomer) Fibrinogen polymerizes to form fibrin network surrounding platelets via thrombin and Factor XIIIa (a=activated) Soluble fibrinogen is the monomer Thrombin (secreted by activated platelets) cleaves fibrinogen at key sites to expose polymerization sites to form fibrin monomer Exposed monomers now form hydrogen bonds with one another to form long fibers that assemble and branch Factor XIIIa and calcium ions promote the formation of strong covalent bonds as fibers cross-link and for the fibrin polymer (aka fibrin network) To prevent blood clot formation, add chelating agens (heparin) to form a complex with calcium ions Forms stable platelet plug which fills the wound area and serves as a scaffold for later repair Scaffold is only temporary; ultimately, the fibrin network is enzymatically broken down during fibrinolysis via plasmin Hemostasis in presence of biomaterial (in contact): platelets often stick to biomaterial and form thrombus by recruiting other platelets and molecules for fibrin network Platelets Little plates 3-4 micrometers in diameter Non-nucleated fragments of megakaryocytes Functions Initially stop or arrest bleeding by adhering to biomaterial surface (if relevant) and/or exposed collagen: platelet adhesion key mediators of adhesion to collagen receptors on platelets: GP1b von willebrand factor (VWF) on collagen conformational changes to integrin receptors on now activated platelets further strengthens adhesion between platelets and collagen promote adhesion to other platelets induce platelet-platelet aggregationHybrid Orbitals Valence shell: the last orbital/shell in an atom to be filled by electrons or the first orbital/shell to lose electrons Valence electrons that occupy valence shell are most likely candidates for participating in bonds and excitation events In several cases where atoms bond to other atoms, the valence shell itself is not a single orbital, but rather a hybrid orbital Sigma and pi bonds sp, sp2, sp3 Pi bonds prevent bond rotation, but are easy to break and excite to a higher energy stateOverview of Fluorescence and Absorption Valence electrons participate in bonding (primary bonds) and in absorption/excitation events Pi bonds are of particular interest for bond formation and excitation Fluorescence Molecular dyeelectronic states Excitation light source at particular excitation wavelength causes electron to jump from ground state to an excited electronic state Nonradioactive decay occurs involving relaxation of electron to a lower, excited electronic state Fluorescence event occurs in which electron relaxes back down to ground state and releases a photon emission For quantum dots comprised of semiconductor materials, typically as size increase, the energy gap decreases (E~1/) Absorption Electron is excited from pi state to pi antibonding state UV-VIS compares relative absorption as a function of wavelength Useful for SEC Useful for determining concentration of macromolecules Peak increases as concentration increasesShort-Range and Long-Range Order in Solids All solids (crystalline or amorphous/noncrystalline) exhibit SRO in which nearest neighbor atoms have a specific spatial arrangement Coordination number is the number ratio of one reference atom t all of its nearest neighboring atoms (relevant for both SRO and LRO) For crystalline materials with LRO, we can assume Periodic arrangement or regular spatial placement of atoms in crystal, crystalline lattice, or lattice A lattice point refers to an individual point (typically one atom) within a crystalline lattice Each lattice point is surrounded by an identical arrangement of neighboring points Typically refers to a single atom whereas a basis refers to a group of atoms associated with a lattice point Unit cell is the smallest group of lattice points that completely describe the structural arrangement of a crystalline lattice Lattice parameters used to describe the structural arrangement of lattice points in a crystal Edge length is the length of an edge in a unit cell Angle between edges Simple cubic, body centered cubic, face centered cubic Close-packed direction: any direction in a unit cell that goes through the centers of touching atoms Close-packed families FCC: {110} BCC: {111} SC: {100} Close-packed planes: 2D cut/plane through the unit cell that includes the centers of atoms, each of which is touching all of its nearest neighboring atoms Close-packed direction and planes are important for calculating atomic packing factor and density of material Volumetric contribution for center is 1, for corner is 1/8, for face-centered is CsCl crystal structure Not a unit cell Not electrically neutral Inner penetrating system of anion and cation NaCl crystal structure Inner penetrating FCCDefects HandoutOverview of Polymers (Handout) Polymer or macromolecule is a chain of molecules linked together by covalent bonds via polymerization Can form new covalent bonds between different functional groups on monomers to form polymers Water is likely to be a byproduct Additional function groups on monomers allow the formation of branched polymers Can break pi bonds in unsaturated (has available pi bonds) monomers to form new covalent bonds in polymer If degree of polymerization, n, is small (2-10), then it is an oligomer (surfactant) If n is large (>>10), then it is a polymer In reality, typically forms many chains with different values for n Be able to draw structures in different notationsConformation vs. Configuration Handout Conformation: the overall shape and structural attributes that can be changed via bond rotation Configuration: structural attributes for polymer that can be changed by breaking bonds For example, breaking bonds to rearrange R groups to change tacticity General structure (polymer architecture) Linear chain: chain with 2 ends Typically can melt from solid to liquid state Chain flexibility, conformation, and crystallinity depend on Concentration of polymer Dilute concentrations: many intrachain interactions possible within same chain High concentrations (heat polymer or polymer melt): both intrachain and interchain interactions and bonds form Chemical composition Steric effects arise depending on the size and placement of side groups Look at rest of handoutFormation of Polymer Networks Utility of heat polymer networks (PMMA) and polymer networks that swell in water (hydrogels) Be able to show electron movement in formation of a linear chain of PAAM with 3 repeat units Be able to show how to cross-link polymer chains to form covalent bonds between chainsCopolymer Up to now, we have discussed polymers comprised of only 1 type of repeat unit to form a homopolymer If more than 1 type of monomer is used during polymerization: copolymer Types of copolymers Random copolymer: AABBBBAABA Alternating copolymer: ABABABAB Block copolymer: AAAABBBBB Self-assembly occurs in presence of water If A was hydrophilic and B was hydrophobic Short chain of A, long chain of B can lead to micelle formation Long chain of A, long chain of B can lead to vesicle formation (commonly used in drug delivery)Step vs. Chain Polymerization Step polymerization Aka condensation polymerization Monomers have 2 or more reactive sites Polymer chains grow step-wise by reactions between ANY 2 reactive species (either monomer and/or reactive chain) Monomer is used up quickly Byproduct often formed (water, HCl) Reaction continues (chain keeps growing) until no reactive groups are left Chain polymerization Aka addition polymerization Monomers usually have the form CH2=CX1X2 Polymer chain grows ONLY by the reaction between a monomer and a growing chain Break unsaturated pi bonds to form new covalent bonds along polymer backbone Usually no byproducts form Involves distinct stages Initiation Creates free radical center initiator species which often has peroxide (-O-O-) or azo (-N=N-) linkage Propagation/growth Typically head to tail addition, but can have head to head addition (sterically hindered) Termination Combination usually results in head to head addition of 2 active chains Disproportionation occurs when one hydrogen atom is extracted from one growing chain by another growing chain Know how to draw polymer products for step and chain polymerization Know how to show electron transfer in chain polymerization Some polymerization routes have aspects of both step and chain polymerization Polyurethanes resemble step polymerization but not byproduct Used in artificial organs One limitation for synthetic polymers is the cytotoxicity of monomers: cant polymerize in presence of cells for cell encapsulation purposes Need to remove any residual monomers Avoid using cytotoxic synthetic monomer (use natural polymer such as alginate or gelatin) Ultimately, its very likely that polymer batch itself will have chains with different molecular weights, resulting from the inability to start and end polymerization at the same time throughout the reaction vessel For a given chain i, the MWi or Mi=n*MWru (Problem Set) Where n is the degree of polymerization, MWru is the molecular weight of the repeat unit, and MWi is the molecular weight of the chain For a polymer chain with many chains, the average molecular weight can be describe with Number average molecular weight, Mn Mn=(NiMi)/Ni Ni is the total number of chains Mn treats all chains equally Weight average molecular weight, Mw Mw=Ni(Mi)2/NiMi Mw permits larger/heavier chains to make a proportionally larger contribution to average molecular weight value In a polydisperse polymer chain in which there is a range of molecular weights, Mw>Mn We indicate the range of molecular weight values in a batch using polydispersity index, PI=Mw/Mn For a monodisperse polymer batch in which all chains have the same molecular weight, PI=1Diffusion In and Out of Biomaterials Diffusion is the movement of atoms or other species through random jumping events In a solid, diffusion requires Free space to jump into (in crystalline materials: vacancies, interstitial, voids) Sufficient thermal energy to overcome activation barrier to jumping or D is the diffusivity (diffusion coefficient), D0 is the material constant independent of temperature, Q is the activation energy barrier, R is the gas constant, T is the temperature Slope is Q/RT and the intercept is lnD0 Though diffusion is a random process, over time, a species will diffuse over same finite distance We can describe the net movement of a group of identical species across an interface to describe a phenomena known as flux Flux is the net number of species that move across an interface or plane of a given unit area per unit time Ficks 1st law J is the flux, D is the diffusivity, and concentration gradient Assuming that concentration gradient doesnt change with time Many biomaterials that deliver therapeutic agents rely on diffusion alone for the therapeutic agent to leave the material matrix PlotCrystallinity in Polymers Considerations Steric effects of side groups: increased bulkiness decreases percent crystallinity Increased chain branching decreases percent crystallinity Tacticity Copolymer: regularity of A and B repeat units Chain folded model to describe semi-crystalline polymers Lamella is the crystalline domain, amorphous is the noncrystalline domain Spherulites are 3D aggregates of lamellae that radiate from a center To create new dislocations or move existing locations, must induce plastic deformation In metals, to decrease the concentration of dislocations, anneal In polymers, to increase percent crystallinity, anneal Anneal: heat (provide energy for defects to move and/or for chains to become more mobile) to an elevated temperature, not to melt the material Slowly cool to allow time for ordering of atoms, chains, etc. If one quickly cools (quenches) the sample, it does not allow adequate time for ordering to occur in material Decreases percent crystallinity, especially in polymersTemperature-dependent Phase Transitions Purely crystalline metals possess clear phase transitions From solid crystal to liquid, melting temperature marks transition Pure liquid phase Viscous response to applied stress Time-dependent deformation response to applied stress is the shear stress, is the viscosity (resistance to flow/deformation), is the shear strain rate Pure solid phase Elastic (reversible) response to small applied stress Independent of time in terms of deformation (assuming that temperature is not elevated) G is the shear elastic modulus Viscoelastic material possesses both solid-like and liquid-like responses to applied stress Characteristic of many polymeric systems G* is the complex shear modulus, G is the shear elastic modulus, G is the shear viscous or loss modulus Pure solid allows us to recover the original structure prior to deformation and recover energy expended into deformation All energy expended into deforming a liquid is lost Possible phase transitions in polymers assuming heating a fresh batch of polymer with no heating history (increase temperature, increase chain mobility) T