Biomaterials Exam II Review Flashcards
1
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Local Biomaterial–Tissue Interactions
A
Effects on material on host tissues versus the effect of the environment on the materials. Examples: infection/tumorigenesis/healing modification/fatigue & corrosion/calcification
2
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Systematic Biomaterial–Tissue Interactions
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Examples: Hypersensitivity, implant elements in the blood, lymphatic particle transport, embolization
3
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Device-Associated Complications
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Thrombosis/infection/bad healing/adverse systemic or local tissue effects and reactions
4
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Provisional Matrix
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Injury to vascular tissue leads to immediate development. Consists of fibrins, produced by activation of coagulation and thrombosis, inflammatory products, activated platelets, inflammatory and endothelial cells
5
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Inflammation
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localized protective reaction of tissue to irritation, injury or infection. Contains, neutralizes or isolates the injury. Cab be acute (first few hours) and chronic (weeks to months). Clinical signs: redness, tissue heating, swelling, and pain
6
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Injury Mechanism
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Vasodilation (redness and heating), increase in permeability leads to exudation (fluid, proteins, blood cells enter the tissue -> swelling), and kinins are released from the blood clotting cascade (pain_
7
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Plasma cells
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Platelets, RBCs, monocytes, PMNs, lymphocytes, basophils, eosinophils, etc.
8
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Polymorphonuclear leukocytes (PMNs)/Granulocytes
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Neutrophils, basophils, eosinophils
9
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Basophils
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Release histamine, heparin, bradykinin, and mediates inflammation, but is the most important in allergic reactions
10
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Eosinophils
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Less phagocytosis, attaches to and destroys parasites
11
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Lymphocytes
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T cells and B cells, apart of adaptive immunity
12
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Neutrophils
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Granular WBC that are the first line of cell defense and are generated in the bone marrow. 4-8 hours in the blood and 4-5 days in target tissue.
They can migrate via:
Rolling -> loose attraction between endothelial cell selection and proteoglycans on neutrophil
Activation -> IL-8 and MIP-1b induce confromational change in integral to have a higher affinity to Ig superfamily CAM
Arrest and adhesion
Transendothelial migration, diapedesis (passage through the walls of the capillaries)
Once they have migrated into the tissues, express high levels of receptors for future chemoattractants to find target area
13
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Phagocytosis
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Neutrophil expresses a greater number of receptors for antibody- and complement-coated foreign particles to help with their removal
Antibody on antigen connects to Fc receptor on the neutrophil, pseudopod surrounds the antigen, making a phagosome. Lysosomes merge with the phagosome, antigen is digests and exocytosis of particles occurs
14
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phagosome
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Vesicle that ingests large particles (antigens)
15
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Phagolysosome
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Fusion of a lysosome and phagosome
16
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Granules
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Contain bactericidal agents and enzymes
17
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Respiratory burst
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Glucose metabolism increases, as well as oxygen consumption. Formation of reactive oxygen and nitrogen species in intracellular granules
18
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MIP-1a and MIP-1b
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Secreted to recruit monocytes (unpolarized macrophages)
19
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IL-8
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Attracts more neutrophils
20
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Monocytes
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Polarize intro macrophages. emigrate from the vasculature and differentiate into long-lived macrophages in the tissue
Cells enlarge, intracellular organelles increase in number and complexity, cells acquire phagocytic ability, and increase soluble factor secretion occurs
21
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Acute Inflammation
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Minutes to days. Exudation of fluid and plasma proteins (edema), emigration and localization of leukocytes at the implant site. Neutrophils get there first. Macrophages dominant, with high phagocytotic abilities. For many biomaterials, they are too big to be engulfed and degraded
22
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Acute inflammation mediators
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IL-1 and TNFa promote inflammatory response (inflammatory migration, clotting) IL-6 and IL-1 are apart of the adaptive immune response
23
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Acute Inflammation systemic response
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fever
24
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Termination of Acute Inflammation
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IL-1Ra produced by the same cells. TGF-b inhibits cell activation involved in the inflammatory response. Acute -> Chronic
25
Cell types in acute vs. chronic inflammation
Neutrophils are high in acute but decrease rapidly in chronic, where in chronic there is a rise of macrophages/FBGCs/fibroblasts, than a later increase in fibrosis
26
In vitro inflammatory response assay
Leukocyte Assays:
-Adhesion -> allow the cell to attach to the testing material for a given amount of time, then rinse. Count the amount of cells that adhered, stain and image and quantify.
-Spreading -> Determine the surface area of the cells
-Migration -> Capillary test test and ring test, Boyden Chamber assay (test cells infiltration into the media of interest through a semi-permeable membrane)
-Cytokine release -> (interleukins, TNFa), using ELISA
-Cell surface markers -> using FACS or flow cytometry
27
Sandwich ELISA
Plate is coated with a capture antibody, add sample antigens present will bind to the capture antibody. Detecting antibody is added and binds to antigen. Enzyme-linked secondary antibody is added. Add substrate, which is converted by the enzyme to a detectable form
28
Flow cytometer
Technique to sort and count particles suspended in a stream of fluid, allowing multi-parametric analysis or physical and chemical characteristics, Uses scattered light to differentiate size and shape of cells and particles. Use fluorescence to detect immunochemically labelled cells or proteins. Sorted via electrostatic deflection, which employs charged plates to change the path of the cell
29
Cytotoxicity Assay
Use established cell lines and positive controls like PVC, gum rubber. Negative controls: high density polyethylene. 3 assays are possible:
Direct contact -> place the test sample directly in the culture, observe under microscope, live/dead stain, MTT assay. Adv: mimics clinical use, Disadv: risk of trauma due to leachable diffusion rate
Agar diffusion
Elution/extract dilution
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Cytotoxicity
Cause toxic effects at the cellular level
31
Agar Diffusion Test
Place sample on an agar plate. The discoloration of the plate expanding from the material indicates the material presence caused cell lysis, losing the stain in the agar layer.
Adv: better concentration gradient and can test one side of a material
Disadv: Flat surface needed, limited by solubility of toxicant in agar, risk of thermal shock and water absorption from agar
32
Elution Test
Determine the cytotoxicity of leachable. Soak the material in MEM and apply extract media at different doses. Good to speratate extraction from testing (test the dose resins), but requires additional time and steps. A positive cytotoxic reaction is seen when the cells lack normal cytoplasmic space and are grainy (indicates high lysis rate)
33
Chronic Inflammation
presence of macrophages, monocytes, and lymphocytes with proliferation of blood vessels and connective tissues.
Lymphocytes are key mediators of adaptive immunity, though little is known about their inflammation response.
Macrophages: most important, releases chemotactic factors, proteases, cytokines, growth factors, etc.
Antigen presenting cells: adaptive immune reactions (B cells)
A short lived chronic response followed by granulation is normal, and persistent chronic ifnallamtion is patholgical and can be triggered by chemical, physical, motion and infection factors
34
Granulation tissue
Forms one day after implantation and seen for about 3-5 days after. Fibroblasts and vascular endothelial cells proliferate and begin to form granule tissues, which is the pink soft appearance on the surface of healing wounds.
Angiogenesis occurs, fibroblasts proliferate and synthesize collagen and proteoglycans, with macrophages present
35
Foreign-Body Reaction (FBR)
Composed of FBGCs and components of granulation tissue (macrophages, fibroblasts, capillaries)
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Foreign body Giant Cells (FBGCs)
Multi-nucleated cells formed by the fusion of macrophages in an attempt to phagocytose foreign materials much larger than a single cell
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FBR topography depends on...
Flat/smooth surface: 1-2 macrophage layer with fibrosis
Rough surface: mixture of macrophages and FBGCs
a higher surface to volume ratio (fabrics, porous materials, microspheres) will have higher macrophage and FBGC ratio to fibrous/granulation tissue
38
Fibrosis/Fibrous Encapsulation
End stage healing response, granulation tissue maturation, larger blood vessels and alignment of collagen fibers in response to local mechanical forces.
The degree if fibrous capsule formation of thickness of the capsule depends on the degree of initial implantation jury (vascular damage, amount and type of subsequent cell death), location of the implant site (low blood vessel density), microstructure of the implant (porosity), amount and composition of the small particulates produced, mechanical factors, implant shape, degradation speed, and electrical current
39
Extrusion
how epithelial cells remove dying or excess cells, squeezing them out without breaking their barrier
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Encapsulation
The typical response to non-resorb able materials
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Resorption
Resorbable material -> Faster degradation limits fibrous capsule formation, slower degradation causes capsule collaspse
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Local Factors on Wound healing Response to Biomaterials and Implants
Site of implantation, blood supply infection potential
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Systemic Factors on Wound healing Response to Biomaterials and Implants
Nutrition, hematologic derangements, glucocortical steroids, preexisting diseases (diabetes, infection, atherosclerosis)
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How to achieve ideal resolution
- Adjust mechanical properties (Reduce stiffness to match soft tissue)
- anti-inflammatory therapy (coatings or local drug delivery)
- Antibacterial treatment
- Surface modifications (roughness/porosity, control protein adsorption, promote cell attachment)
- Implant size/geometry (avoid sharp edges and corners
- Minimize motion and toxic release
- Reduce degradation (wear debris, corroded metal ions etc.)
- Bioactive bonding (eg. bioglass)
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In Vivo Assays: Animals
Use species similar in physiology and healing response to humans, start small (rodents, rabbits) then move to large (goat, dog, sheep, cow)
The implant sire should be close to application site, but can use somewhere more accessible at the start
The study should have multiple time points.
46
In Vivo Assessments
Histology:
- Fix (crosslink with aldehydes, precipitate with alcohols, acetone, acetic acid, add oxidizing agents such as osmium tetroxide)
- Section with cytostat or paraffin embedding & microtome. Resin embedding ultra-microtome sectioning
- Stain with IHC or H&E
- Optical or Fluorescent Imaging
- Quantify
Electron microscopy for ultra thin sections
Biochemical assays: western, RT-PCR
47
Coagulation
Hemostatic mechanism to arrest bleeding from injured blood vessels: vascular contraction, platelet plug, blood coagulation (thrombosis)
Similar process produces adverse effects when artificial surfaces come in contact with the blood.
Key players: coagulation proteins, platelets, and endothelial lining of the blood vessel
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Blood coagulation
complex set of interdependent reactions between the surface, platelets and coagulation proteins resulting in the formation of a clot or thrombus that may undergo removal by fibrinolysis, localized process at the surface
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RBCs in Blood Coagulation
Usually passive, under low shear or venous flow, they may form large proportion of the total thrombus mass.
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WBCs in Blood Coagulation
Activation of complement coagulation and fibrinolytic and other enzyme system
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Platelets in Blood Coagulation
Arrest bleeding through the platelet plug, stabilize this plug via catalyzing coagulation reactions (fibrin formation)
52
Platelets
Activate via adhesion (activate via interactions between cell surface receptors and ligands on collagen, vWF, fibrinogen, and fibronectin), adhesion to the damaged endothelial wall (Collagen) or biomaterial surface (plasma proteins)
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Platelet activation cascade
Activate -> become discoid shape (irregular shape with tiny pseudopods) -> contraction of cytoskeleton proteins causes thrombin, ADP and thromboxane 2 release -> stimulates even more platelet activation -> Glycoprotein receptor GP IIb/IIIa activation causes binding to plasma proteins (fibrinogen-vWF) or platelet-platelet binding leading to aggregation -> catalyzed coagulation with Factor X activation and a membrane forms catalytic environment to turn prothrombin to thrombin
54
Coagulation Cascade Intrinsic System
Can be triggered by lipid flipping.
Factor XII ––(neg. charged surface contact)––> XIIa
Factor XI ––(XIIa)––> XIa
Factor IX ––(XIIa, Ca++)––> IXa
.....
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Coagulation Cascase Extrinsic System
Factor VII ––> VIIa
.....
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Coagulation Common Pathway
Factor X ––(IXa, Ca++, Factor VIII, platelets) ––> Xa
Prothrombin ––(Xa, Ca++, Factor V, Platelets) ––> Thrombin
Factor XIII ––(thrombin)––> XIIIa
Fribinogen monomer ––(thrombin)––> fibrin (polymer)
fibrin –––(XIIIa, Ca++)––> stable fibrin
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Fibrin Polymerization
Fibrinogen + thrombin removes fibrinopeptides A and B, forming fibrin monomer which then dimerizes before polymerizing
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Blood Clot Primary cells
Blood cells, platelets, fibrin clot
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How to limit clot formation
Dilute blood flow, rate of clotting is fast only when the reaction is catalyzed by a surface (conversion of X to Xa, which controls prothrombin to thrombin)
Inhibit thrombin/coagulation enzymes (heparin, anti-thrombin III (ATIII) complex), thrombomodulin
Enzymes that activate coagulation factors and degrade cofactors
60
Fibrinolysis
Remove unwanted fibrin deposits to improve blood flow after thrombus formation to facilitate healing post-injury and inflammation
Plasminogen -> plasmin (Factor XIa, XIIa, tPA, urokinase) breaks fibrin up
61
Complications of Blood-Material Interaction
- Prolonged cardiopulmonary bypass and membrane oxygenation can produce neuropenia
- Mechanical heart valves shed emboli (detached blood clot that can travel through the bloodstream and lodges to obstruct/occlude a blood vessel) leading to stroke
- Graft failures due to thrombosis -> ischemic and death of downstream tissue beds
- Systemic anti-coagulant administration leads ti bleeding risk
62
Adhesion proteins in plasma for platelets
include fibrinogen, fibronectin, vitronectin, and von Willebrand factor
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Platelets are ___________ to adsorbed plasma proteins, one solution is to create a ____________ surface
(Sensitive, non-fouling)
Receptors IIb/IIIa and Ib/IX bind to adborped proteins and mediate adhesion
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Non fouling surface
Anti-adhesion surface modifications or properties
65
Role of the endothelium
Solution based on this?
At rest, anti-coagulative properties: glycocalyx, heparin sulfate aids ATIII to inhibit thrombin, thrombomodulin on EC surface binds to thrombin, activates protein C to inhibit coagulation. Secretes soluble chemical mediators that prevent aggregation or active plasmin, promoting fibrinolysis
Post-injury...
The glycocalyx is compromised, the now exposed ECM is reactive to plasma proteins and platelets, and in response to pro-inflammatory cytokines, the intact Ec surface may become coagulator (reduce thrombomodulin, secrete TF and vWF)
SOLUTION? drug eluting material, or dip in thrombomodulin, graft in heparin sulfate or ATIII.
Or, try endothelial cell sending, as the endothelial vessel can maintain latency for a lot longer (but this is difficult to culture and difficult to seed into the graft)
66
Static BMI In vitro Assays
Place whole blood (non anticoagulated or anticoagulated with sodium citrate) into containers made of test material, control: glass
Measure clotting time, phenotypes of adhered platelets, mass of thrombus, amount and types of platelet release or other clotting factors
67
Dynamic BMI in vitro Assays
Circulating heparinized or citrated blood through a tubular device made of the test materials
Measure clotting time, phenotypes of adhered platelets, mass of thrombus, amount and types of platelet release or other clotting factors
68
Blood
Cells and Liquid
69
Plasma
Liquid remaining after anticoagulant has been added to the blood, no cells
70
Serum
Liquid that remains after the blood is clotting, no cells
71
Natural vascular grafts
Use the saphenous vein, complete endothelization, symptoms can reappear, mismatch mechanical properties, and its not always feasible
72
Synthetic vascular grafts
Porosity enhances healing, impregnate with connective tissue proteins to help with clotting, reduce blood loss through pores, stimulate tissue ingrowth/antibiotic release.
Pre-clotting.
Good in large diameter high flow, has low resistance locations
Small diameter is more challenging
73
Tissue engineering for vascular grafts
Used when a small diameter is needed. Scaffolds with cell seeding, or a cell free scaffold that is degradable/decellularized. This however needs to achieve stable and complete endothelialization
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Vascular graft healing
Fibrinogen adsorption, pseudointima, smooth muscle integration and proliferation. Endothelial cell lining forms near the suture, the rest covered by pseudointima, this can be dislodged
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Basic Mechanisms of the Innate Response
Phagocytes encounter and ingest the microbe. They degrade by releasing ROS intermediates and proteases. Release cytokines to attract more cells to the site. Activate adaptive immune cells
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large biomaterial > phagocyte
Macrophages fuse to FBGC, release ROSs and proteases into environment in attempted to breakdown material
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Complement activation
Activation of a complement by biomaterials leads to inflammatory cell accumulation, leads to failure.
C3 adsorption -> macrophage adhesion
C3b attachment of biomaterial -> opsonize foreign objects, make them more likely to undergo phagocytosis
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Humoral Adaptive Immunity
Antibodies from B lymphocytes mediate. Memory B cells express membrane bound antibodies. Plasma cells produce the antibodies. B cells activate by antigen binding and a stimulatory signal from Th cells into plasma and memory
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Antigen
Substance that binds to antibodies/TCRs to innate acquired immune response. The site recognized by the antibody is an epitope, one antigen may have multiple epitopes
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Antibody
Protein secreted by B cells with high antigen specificity. 2 light, 2 heavy chains.
Fab fragment – variable portion recognized by antigen
Fc fragment – constant, recognized by phagocytes or complements
IgG, IgD, IgE, IgA, and IgM
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T lymphocyes
Th (helper t) provide signals and cytokines to orchestrate cellular activity
Tc (Cytotoxic t) kill selected targets. Target INTRACELLULAR pathogens
T cells cannot recognize proteins unless that have been degraded into small fragments and bound onto APCs
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Antigen presenting cells
Presents antigens and activate T cells. Macrophages, B cells, dendritic cells and langerhans cells
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Major Histocompatibility Complex
Antigen must be displayed with MHC I or II.
Different person to person. MHCs not recognized by host with elicit T cell activation, B cell antibody production -> graft destruction, failure.
Avoid via: tissue typing, immunosuppressive agents
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MHC I
Transmembrane glycoprotein found on all nucleated cells with B-macroglobulin. Intracellular antigens are degraded and presented with MHC1 to Tc cells
85
MHCII
Only on APCs. Extracellular antigens ingested and presented to Th cells, activates macrophages or B cells
86
Origin of Specificity/Memory
B cells produce antibodies specific to antigens. T cells produce TCRs. antibodies/TCRs expose to self-antigens and undergo apoptosis (self vs non self recognition)
After activated, lymphocytes rapidly divide, specific antigen population
87
Immune Response to Biomaterials
Synthetic biomaterials -> no specific immune response, can have failure modes. Still can activate innate
Decellularized tissue or collagen-based biomaterial do not normally cause adaptive immune response. No cells = no foreign MHCs, proteins are similar.
Denatured native proteins mya have undesired immune response
88
Hypersensitivity/Allergy
Hypersensitivity: the immune system strikes invading molecules with high intensity towards the relative amount of damage done
Allergen: foreign substance. Large foreign molecules (proteins and nucleoproteins) are strong, lipids are not
89
Hapten
Low MW substance combining with carrier molecules to create greater immune response (synergy). Second exposure - antibodies react against happen without carrier (metal allergy, Cp, Cr, Mo. Methyl methacrylate resin, accelerators/antioxidants in latex)
90
Type I Allergy
Plasma cells secrete IgE -> IgE binds to mast cells, basophils -> sensitization
Second exposure -> crosslink membrane bound antibodies -> mast cell degranulation (release of mediators such as histamine, vasoactive substances) -> vasodilation, smooth muscle contraction
91
Type IV Allergy
Cell mediated, more than 12 hours (24-7). Previously sensitized T cells -> release inflammatory mediators -> tissue damage both at and away from exposure site
92
Tumorigenesis
Neoplasia, new growth, unregulated process of excessive/uncontrolled cell proliferationTm
93
ors
proliferating neoplastic cells and supportive stoma of connective tissue and blood vessels
94
Metastasis
Development of secondary malignant growth distances from the primary site
95
Carcinogen
Stimulus causing malignant transformation vs mutagenesis
96
Malignant Transformation Process
Accumulation of genetic damage.
Imitation -> latency -> promotion
97
Biomaterial Tumorigenesis
Tumorsassociated with clinical implants in humans are rare, hard to demonstrate causality.
Chemical carcinogens–leachable or degradation products from polymers can be carcinogens, but no evidence shows they cause tumor formation at relevant dosages
98
Foreign Body Carcinogenesis
Small fibers: mesothelioma
For large implants, tumorigenesis and correspond directly to the extent and maturity of tissue encapsulation.
Implants with continuous surface area: more tumorigenic
Fabrics/fibers produce fewer tumors than sheets of the same materials, and powers produce almost no tumors
99
Foreign Body Tumorigenesis
Cellular FBR -> fibrous capsule forms -> preneoplastic cells contact the surface implant during tissue reaction -> preneoplastic cell maturation and proliferation -> tumor growth
100
Carcinogenicity Test
Should be conduced if data from other sources suggest tumor induction tendency
In vitro -> ames test
In vivo -> long term rodent study with positive and negative controls, harvest at different time points, analyze with SEM and histo
101
Ames Test
Carcinogenicity in vitro test. Evaluate mutagenic potential, requires histidine to grow but lacks ability to biosynthesize it because of essential gene mutation
102
Infection
Invasion by and multiplication of microorganisms
103
Gram positive bacteria
Strong well well with telchoic acid and peptidoglycan
104
Gram negative bacteria
LPS, can trigger the inflammatory response
105
Biofilm
Bacterial adhesion to surface in aqueous environment, excrete glue-like substance hat anchors them to different materials surfaces.
Can be one or many species of bacteria, as well as fungi, algae, protozoa, debris, and corrosion products
Found on all failed medical devices
106
Infection steps
Bacterial attachment (VdWs, reversible)
Bacterial adhesion (ligand-receptor binding, irreversible)
Aggregation -> biofilm formation. Change phenotypes, produce matrix, proliferate and recruit (form micro colony), mature and into thick and stable structure
Dispersion -> spreads the infection
107
How do implants facilitate infection
Provide access to circulation and deeper tissue by damaging natural barriers against infection
Provide surfaces for bacteria to attach to
Limit phagocyte migration to infected tissue or interfere with inflammatory cell
108
Factors influencing rate/extent of biofilm formation on biomaterials
Microorganism exposure. Microorganisms must adhere, cell attacghemtn rate depends on: number/types of cells in liquid, flow rate, physiochemical surface characteristics components in liquid altering surface properties.
Once they attach and product extracellular polysaccharides, bacteria don't attach to the surface but to slime. Biofilm growth rate is influenced by (flow rate, nutrient composition, antimicrobial-drug concentration, and temperature)
109
Biofilm control
non-fouling surface is effective.
Agents: antibodies, planktonic phenotype lock in (signal blockers)
Dc field, ultrasonic energy
Delivery of agents to kill planktonic (systemic injection, irrigation after installation, local release from surface)
110
Sterilization Methods
moist heat: saturated steam, destroy metabolic/structural component of microorganisms. Good for metallic/heat resistant surgical instruments. High speed, simplicity, efficacy, and no toxicity. Bad b/c high temp, pressure degrade product/packaging materials
ETO: toxic, carcinogen, remove residue after 2-16 hours. Alkylation of amine group. High efficacy, high penetration ability, high compatibility. Bad b/c residues
Radiation: Gamma ray, ionization causes microorganism death. simple rapid effective, but high cost and can be incompatible
111
In vitro infection models
determine if biomaterial encourages/discourages bacterial attachment. Analyze the source of infection
112
Ex vivo and in vivo infection models
bacterial may be directly places at implant site or via injection.
Ex vivo is good for blood contact devices
Markers of infection: histo, SEM, leukocyte/lymphocyte counts in blood. Measures the amount of bacterial cell wall antibodies found in blood. presence of LPS
113
Calcification
Formation of nodular deposits of calcium phosphate or other calcium containing compounds. Used for osteoinductive materials used for orthopedic/dental applications. undesired for non skeletal tissue replacement and devices that aren't supposed to calcify -> mech failure
Often principle sites are dead cells and cell membrane fragments
Affect both tissue derived biomaterials and synthetic polymers
114
Biological heart valve xenograft
Porcine aortic valve/bovine pericardium. Fix with glutaraldehyde to preserve tissue and kill cells to reduce immunogenicity
115
Biological heart valve allograft
human cadaveric aortic or pulmonary valves with/without vascular conduits, good hemodynamics, low thromboembolic complications and low infection. Cryopreserved, low thrombosis rate. Less durable, structural damage due to tissue deterioration
116
Calcification
Tear and stiffening (stenosis), intrinsic on cusps. Extrinsic (in thrombi or endocarditic vegetations), accelerated in youth
117
Factors causing calcification
mechanical force -> cell death/deformation
Fixing agents like glutaldehyde, stabilize cell surface proteins
Nonviable cells lost mechanism to remove Ca++
Phosphate contains proteins on cell membrane act as a nucleation site
alkaline phosphatase on membrane promote calcification
Collagen act as a template for growth of mineral crystals
118
Calcification prevention
Systemic treatment of local delivery of anti-calcification agents (HA formation inhibitors, bisphosphonates, trivalent metal ions from complex, calcium diffusion inhibitor, 2-amino oleic acid covalently binds to glutaldehyde, diminishes Ca++ diffusion)
Biomaterial modifications, removal of calcifiable component, addition of exogenous agents/chemical alteration
-surfactants: remove phosphate containing proteins/acidic phospholipids
-ethanol: extract lipids, alter collagen conformation, affect cusp interactions with water/lipids
-decellularization
Modification of glutaraldehyde fixation or use other cross-linking agents
119
Calcification assessment in vitro
physiologically relevant media with chemical composition mimicking urine or blood or CSF. Static or dynamic
120
Calcification assessment in vivo
Subcutaneous: easy access, faster calcification. Final location
121
Other calcification assessments
Chemical assays to determine Ca or phosphate content
Sectioned and stained for Ca content
Explant analyzed by SEM, SEM-EDAX, X-ray diffraction
In situ imaging: radiography suing X-ray, microCT
122
Bioelectrode Application
Conducts current across the interface between the body and the electronic measuring circuit. Serves as a transducer to change an ionic current into electronic current and vise versa
Recording electrodes monito electrical events in the body: ECGs, EEGs, EMGS,a nd BMI
123
Stimulating electrodes
Transmit electrical current into the bd=ody to influence specific biological processes (cardiac pacemakers, defibrillators, deep brain stimulators, transcutaneous drug delivery), bioelectronic medicine, electrochemical biosensors
124
Faradaic process
Charge transfer governed by faraday's law (the amount of change occurring at the electrode-electrolyte interface is directly proportional to the current that flows through the interface. Electrodes that undergo mainly faraday processes are called charge tarsier electrodes/non-polarizable electrodes
125
Polarization at the elctrode-electrolyte interface
the half cell potential of the electrode results in the formation of an electrical double layer, which can be considered as a capacitor
Perfectly polarizable electrodes do not undergo faraday processes (e.g., noble metal Pt, Au, stainless steel)
126
Non-faradaic process
current flow can be passed by charging and discharging of the capacitor
127
Mixed Situation (faraday)
Faradaic and nonfaradaic pathways of general electrode reactions. The charge transfer reaction at the surface is faraday and the diffusion and thew adsorption/desorption processes are nonfaradaic
128
Faraday circuit model
battery-like Ehc is the half-cell potential. Rd and Cd ate impedance and polarization effects in parallel, and Rs is the series resistance associated with interface effects and due to electrolyte resistance
129
Impedance Spectroscopy
Z(w) = V(w) / I(w) = |Z(w)|e^(iø)
|Z(w)| = [ReZ(w))^2 + ImZ(w))^2]^(1/2)
130
Non-polarizable electrodes
Ag/AgCl
Ag <––> Ag+ + e-
Ag+ + Cl- <––> AgCl (precipitate).
Can be made by electrolytic processes or entering processes
131
Skin Surface Electrodes
Electrode -> Gel -> epidermis ->dermis and subcutaneous layers.
Needs to overcome the stratum corneum layer
132
Microelectrode assays (MEAs) for neural recording include...
Utah array, microwave array, Michigan probe
133
Michigan Probe
Gold pads contacting instrumentation -> polycrystalline silicon -> gold or iridium electrode sites
134
Cellular Response around inserted Neural Electrodes
Macrophages.microglia.astrocyte/enurons. After awhile, the astrocytes and microglia bind to the polycrystalline silicon
135
Electrode modification using conductive polymers
3,4-ethylenedioxythiophene + A- –––(-e-)–––> PEDOT
A is proteins/peptides/anti-inflammatory neuroprotective, neurotrophic factors and neurochemicals
136
PEDOT/PSS facilitate charge transfer at electrode/tissue interface
Low impedance. Enables high quality recording from ultra-small sites
137
Stimulating electrodes
produce transient electric fields in their vin city to modulate electrophysiological actives.
The uniformity and precision of the e- fields are dependent on the charge density distribution created by the stimulating electrodes
138
Charge injection limit
Maximum quantify of charge that can be injected per unit area.
Biphasic current pulse: negative then positive step function.
Voltage Response: First: instantaneous drop, -E.mc, then there is a polarization curve (V.access), monitor voltage electrode experiences, this curve is where polarization occurs.
Goes back up then over.
Etc: maximum cathodic potential, Ema: maximum anodic potential
Safe potential window (water window): -0.6–0.8V.
Q/A = Ixt/A
139
Stimulation Electrodes
Noble metals: mostly use noble metals which undergo minimal chemical reactions (Pt is stable, inert. Pt/Ir allow is harder. Ir and iridium oxide is reversible reactivity
Non-noble metals: stainless steel or Ni/Co based alloy, susceptible to corrosion related failure
Stimulation waveforms: voltage stimulation is susceptible to complex fluctuations in comparison to current stimulation (cannot go above the safety limit)
Pulsatile current stimulation is charged delivered = current x duration
Biphasic pulses are better than monophonic pulses: minimize tissue damage by reducing the irreversible electrochemical reaction
(can be asymmetric with a higher negative side and longer, longer positive side)
140
Cardiac Pacemakers
For patients with cardiac arrhythmias. Deliver current to myocardium via electrodes, resulting in depolarization/heart contraction. Senses intrinsic cardiac activity
Active electrode fixation to endocardium – designed to grasp the surface of the heart
Passive electrode fixation to endocardium – addition of projection tines/fins
Porous metal surface to encourage tissue ingrowth
141
Stimulation threshold
minimum voltage required to stimulate the heart outside of the refractory period. this threshold can increase, and the battery of the pacemaker by drain as the voltage increases
142
Electrode scarring
Inflammation results in fibrous capsule, increases the threshold. This can be reduced with improved design and a local release of corticosteroid.
Conventional actively fixable pacemaker lead electrode with a screw on the distal end versus a lead electrolyte with additional steroid eluting ring for reduced trauma
143
Natural protein polymers
Collagen, gelatin (partially amorphous collagen), fibrin/fibrinogen, silk (silkworm/spider)
144
Natural polysaccharide polymers
Cellulose, carboxymethylcellulose, methylcellulose, alginate, GAGs (heparin, HA, chondroitin sulfate), Chitin/chitosan
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Natural polynucleotide polymers
DNA, RNA
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Natural polymer polyester made by bacteria
PHB/PHV polyhydroxybutyrate/polyhydroxyvalerate
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Natural vs synthetic biomaterials
Natural has high biocompability with biological tissues.
Synethic materials usually have no immunogenicity but could produce some if there is an allergy. Natural materials might have immunogenicity
Molecular weight of natural is defined, despaired in synthetic
Few natural polyhedral processing choices compared to synthetic
Natural has low reproducibility compared to synethctic
Natural degradation is safe, synthetic may or may not be
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Collagen
Most abundant natural polymer. 10 different types, differ in length and carbohydrates attached.
Type I: skin/tendon/bone, 90% of collagen. in scar tissue (OA bad)
Type II: cartilage (OA good)
Type III: major component of blood vessels, also in skin and granulation tissue prior to type I formation
Type IV: component of basement membrane separating epithelial tissue from mesodermal tissue
Type I-III forms fibrils with distinct triple helical structure while type IV is non helical, no fibril formation
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Type I Collagen Structure
Primary structureL animo acid sequence along peptide chain (Gly-X-Y), where X and Y are often proline and hydroxyproline
Interchain crosslinks are the condensation of lysine and hydroxylysine
Secondary structure is an alpha chain
Tertiary structure is a triple helical structure held by H-bonds
Quaternary Structure (super molecular unit structure): several triple helical molecules packed in quasi-hexagonal lattice-> microfibrils->lateral and end-to-end aggression-> fibril-> collagen fiber -> bundled fibers lead to body tissue formation
more interchain crosslinks as we grow old
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Importance and Manipulation of Structural characteristics
2 discrete levels of structural orders: 3° and 4°
Helix heating -> random coil like gelatin and fast degradation
D-period -> platelet aggregation (thrombogenic)
Can decrease the thrombogenecity with treatment
D-period -> acetic acid (pH<4.25)-> D band disappears and helix is unchanged
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Chemical Modifications: Degradation
Collagenases are naturally occurring enzymes that attack the triple helix at a specific location 2/3N, 1/3C => gelatin
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Chemical Modifications: Cross-linking
NH2 on lysine residue can be cross-linked with dialdehydes. The crosslinking reduces degradation rates from days to weeks. Cross-linking decreases immunogenicity problems
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Chemical Modifications: enzymatic treatment
Cleaves telopeptide region and may reduce the immunogenicity
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Chemical Modifications: gelatinization
Increases the immunogenicity
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Collagen Applications
hemostatic devices such as gel form (water-soluble, absorb/hold within interstices many times its weight, can be applied to bleeding surfaces)
Lipid augmentation, nerve conduits, tendons/ligaments, burn treatments/skin grafts, drug delivery, heart valves
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Fibrin Gels
Formed from fibrinogen and thrombin. The fee structure can be modified and controlled based on Ca++ and fribinopen concentration
Amenable to inclusion of growth factors
Degraded by plasmin, degradation products are digested by inflammatory cells (applications in tissue engineering and sealent)
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Sealent
2-component fibrin mixture that simulates natural physiological conditions to achieve hemostasis and tissue sealing
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Hyaluronic Acid (HA)
Native ECM sugar, high content in epithelial, connective and neural tissue.
Linear polysaccharide with repeated units. Ranges in size
only insulated GAG.
Hyaluronan is degraded by a family of enzymes called hyaluronidases
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HA Applications
hydrophilic, anti-inflammatory and pro-regeneration abilities. Adhesion barrier, ophthalmology, soft-tissue implants, wound healing, surface coatings, drug delivery, cosmetic applications, OA knees
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Chitin
Found incrabs/shrimps/insects/worms.
Tough, strong, used in sutures and wound healing agents
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Chitosan
Obtained by deacetylation of chitin and is more water soluble.
Used for gene deliveries, and bandages
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Polyhydroxyalkanoates (PHAs)
Aliphatic polyesters naturally produced via a microbial process on sugar or lipid based medium. where they act as carbon/energy storage material in bacteria), can be thermoplastic of elastomeric materials
PHB: poly-beta-hydroxybutyrate/ PHV: polyhydroxyvalerate.
Completely bioresorbable, no toxic byproducts, solution for plastic waste issue. Being examines as biomaterial for bone repair, fixation, suture, and tissue engineering scaffolds
