Lecture 4 Flashcards
The first steps of biomaterial-host interactions
- Water and ions (quasi-instantaneous)
- Proteins, arrive rapidly (~1s) by diffusion or convection
- Cells reach the surface and the adsorbed proteins.
• adhesion/non-adhesion
• activation/inhibition of cell activity
• internalization (for small objects)
Protein adsorption and host response
Adsorption of (adhesion) proteins provides recognition sites for cell integrins at the surface of biomaterials
Controlling protein adsorption: a key to modulate host response.
Coatings of cross-linked phosphorylcholine (PC) reduce protein adsorption and cellular adhesion -> reduce thrombus formation
Protein adsorption at solid-liquid interfaces
Kinetics of adsorption:
1- Rapid phase in t(1/2) diffusion-controlled process
2- Slower phase where protein adsorption is hindered
(Graph shows rapid growth and then plateau)
Adsorbed proteins usually bind ___
tightly to the surface.
Surface physical-chemistry of material ___
affects the conformation of adsorbed proteins. (Ex: Hydrophilic surface binds with different side chains than hydrophobic)
Conformation changes
alter biological activity of adsorbed proteins. Ex: Variation of the platelet binding to fibrinogen
Degree of denaturation highly depends on:
- protein type
- surface physical-chemistry
- time spent on the substrate
- solvent
In complex mixtures, composition of adsorbed protein layer results
from
differences in affinity and in relative concentration
The Vroman effect
When a surface is placed in contact with a mixture of proteins, the highest mobility proteins generally arrive first and are later replaced by less motile proteins that have a higher affinity for the surface.
Injury/implantation induces bleeding
-> Hemostatic mechanisms to arrest bleeding
Hemostatic process
Interdependent reactions between: 12 coagulation proteins & platelets
Platelets
(0.3% blood volume) with size of 3microns
Platelet in normal (non-active) state
are maintained in discoïd shape by circumferential bundles of microtubules
External surface of platelets is coated with
Membrane bound receptors Glycoproteins GPIb (25,000/plt) and GPIIb/IIIa (80,000/plt). Their membrane also form a spongy canalicular structure. Large reactive surface.
Several important molecules for coagulation are stored in cytoplasmic granules
- dense granules (ADP, Ca2+, serotonin)
- a-granules (PF4, plasma proteins,…)
- lysosomal granules (enzymes acid hydrolases…)
Platelet activation
Platelets respond to minimal stimulation.
Activated platelets:
- change shape
- become sticky
- release from the granules (coagulation factors, adhesion molecules…)
Coagulation is __
The result of a complex cascade of activations of factors (proteins). 3 main phases lead to the polymerization of a network of fibrin chains including platelets, forming the blood clot (Thrombus)
Intrinsic pathway
Activated by negatively charged surfaces (surface of activated platelets, collagen, glass…)
It is intrinsic because triggering factors are in the blood itself.
Slower than extrinsic due to numerous intermediate steps.
Extrinsic pathway
Activated by exposure of blood to factors located below endothelial tissues (e.g. after an injury): the tissue factor (III).
It is extrinsic because triggering factors are out of the blood.
Fast (few steps): allows clot formation in 15sec.
What is phase 2 of coagulation?
The plasmatic protein Prothrombin is transformed into Thrombin (enzyme)
What is phase 3 of coagulation?
Thrombin catalyzes fibrin polymerization Activated Factor XIII stabilizes the clot
What prevents coagulation from spreading ? Several mechanisms avoid massive thrombus formation:
- Blood flow reduces localized concentration, dilution and removal through the liver.
- Several reactions are catalyzed near a surface (need the proximity of tissue factor or activated platelets)
- Blood plasma contains anticoagulant factors.
- Platelets and endothelial cells also release anticoagulant factors upon activation (plasmin, factor C)
End of hemostasis
Fibrinolysis: In normal in vivo situations, clot formation ends 3-6min after vessel injury. After 30-60 min, clot contraction and closing of blood vessels. After 2 days, dissolution of the clot by fibrinolytic enzymes (plasmin…)
When artificial surfaces are in contact with blood,
hemostatic mechanisms may have adverse consequences. Ex: Metallic coronary artery stent -> thrombosis
Hemocompatibility
Ability of a material or device to perform its proper function in contact with blood, without eliciting adverse reactions (thrombus,…)
Blood coagulation is governed by three factors:
- blood chemistry
- blood-contacting surface
- flow regime
Use of blood-compatible materials (non-thrombogenic)
does not guarantee blood-compatible device.
Intrinsic pathway to coagulation via implant
- Modification of surface by protein adsorption
- Platelet adhesion
- Desorption of protein fragments and denatured proteins -> platelet activation
- Activation of coag. factors (esp. XII) on material surfaces
Extrinsic pathway to coagulation via implant
Possible lesions during implantation
Perturbation o fcoag/anticoag balance via implant (synthetic surfaces do not produce and release coag. inhibitors like endothelial cells of blood vessels)
• Flow perturbation (low shear or static regions)
Improper blood-implant interactions (esp. thrombosis) continue to limit the potential of many cardiovascular devices for applications in human.
- Cardiopulmonary bypass for oxygenation -> severe bleeding tendency
- Mechanical heart valves -> emboli -> stroke
- Synthetic vascular grafts -> thrombosis -> ischemia and death of downstream tissues
- No existing grafts for small diameter (<4mm) vessels
Most often, systemic anticoagulants are needed in addition to the device, but…
This leads to inherent bleeding risk
Functions of blood clot
- Barrier to infection
- Arrest of blood flow and exudation
- Reservoir of chemo-attractants mitogens cytokines growth factors
The effect of an injury to a tissue:
• Changes in vascular flow
• Escape of fluid, proteins, cells into the injured site (Exudation)
Activates cellular events (inflammantion)
Host response to implantation of device (Almost the same as wound healing)
- Injury
- Blood-material interactions
- Provisional matrix formation
- Acute/Chronic inflammation
- Granulation tissue
- Foreign-body reaction
- Fibrosis/fibrous encapsulation
Protein adsorption leads to
Activation of
- coagulation cascade
- complement system
- platelets
- immune cells
What is the complement system?
a group of blood plasma proteins (~30) that can be activated in cascade to stimulate cells of the innate immune system. (IgG, C3)
Steps of inflammatory response
- Protein adsorption: blood coagulation and immune system activation
- Release of danger signals (alarmins or DAMPS)
Acute inflammation
Duration: minutes to days depending on extent of injury normally resolves quickly (<1week) if not, suggests an infection.
Characteristic component: Neutrophils (PMN: PolyMorphoNuclear leukocytes)
Major role of PMN
Clean the place. The phagocytic response.
3-step process of phagocytes
1- Recognition and attachment 2- Engulfment 3- Killing or degradation
PMN are degraded by release of :
- Proteolytic enzymes (proteases)
- Reactive Oxygen Species (ROS)
Consequences of PMN degradation
Material corrosion
Damage to surrounding tissues
-Prolonged inflammatory response Exhaustion of PMN killing capacity
-Risk of severe biomat-centered infection
Resolution
< 2 days after implantation PMNs disappear from implantation site.
Chronic inflammation develops as
inflammatory stimuli persist at the implant site.
Chronic inflammation
Characteristic components:
Macrophages / Monocytes Lymphocytes + proliferation of blood vessels and connective tissue.
Activated by inflammatory stimuli biomaterial-related activation depends on:
• chemical and physical properties of the biomaterial
• adsorbed protein layer at biomaterial’s surface
• motion of the implant site
• infection
One of the main actors of chronic inflammation?
Macrophages
Marcophage characteristics
- Phagocytosis
- Produce many biologically active products - neutral proteases - chemotactic factors - coagulation factors - growth promoting factors - cytokines - …
- Stimulate migration, differentiation of other cells for formation of granulation tissue.
Macrophages can take on 2 different states
M1 (classically activated phenotype) Killing! Inflammation!
M2 (alternatively activated phenotype)
Phagocytosis of wound debris
Promotion of wound-healing Limitation of inflammation. Repair :) Anti-inflammatory :)
The granulation tissue
- Within 1 day, proliferation of fibroblasts and endothelial cells to form the granulation tissue.
- Formation ~3-5 days following implantation.
When a biomaterial is >5μm
not phagocytable, leads to formation of foreign body giant cells
Foreign Body Reaction characteristics
Characteristic components:
• Foreign Body Giant Cells formed by fusion of macrophages.
• Components of the granulation tissue
(macrophages, fibroblasts, capillaries…)
FBR depends on the form and topography of the foreign body
• Flat and smooth surfaces
(e.g. silicone breast implants)
Layer of macrophages (1-2 cells thick)
• Rough surfaces
Macrophages
+ FBGC
• Fabric materials
Macrophages
+ FBGC + granulation tissue
Putative mechanism of FBR
Important release of proteases by FBGC
• Alter surrounding tissues
• Activate fibrosis
Design of materials with “immunomodulating” capacities
Passive way: By physico-chemical modification of surface
Active way: By using molecules or matrices to target specific cell behavior.
Immuno-modulation by surface modification
General approach: Reduce macrophage adhesion, activation, fusion to FBGC
Rat cage implant example of PASSIVE immuno-modulation by surface modification
On hydrophilic and anionic surfaces
DECREASE macrophage adhesion and fusion INCREASE macrophage apoptosis
ACTIVE Immuno-modulation using bioactive molecules
- Providing integrin adhesion sites
- Grafting/Release of anti-inflammatory drugs
- Grafting/Release of growth factors
Providing integrin adhesion sites
Short oligonucleotide sequences can be recognized by cell receptors (integrins)
Grafting/release of anti-inflammatory drugs
Nitric Oxide (NO) is a natural mediator in wound healing and angiogenesis
Grafting/Release of growth factors
Growth factors stimulate tissues/cells to modulate macrophages. Generally successful but need of non-physiologically high amounts (expensive, possible side effects)
What is the idea of immuno-modulation using ECM coating
mimic the regulating properties of ECM
What is infection?
The process of invasion and multiplication of pathogenic microorganisms such as bacteria, viruses, and parasites within the body
The number of device-related infections is increasing due to:
- Longer lifetimes and implantation durations.
* Increasing number of invasive surgical operations
Bacterial biofilms
are communities of bacteria which attach and grow on surfaces of abiotic materials or soft tissues
Problematic bacterial infections by staph
- Propensity to form biofilms.
* INCREASE of multi drug resistant forms
Infection routes w/ implant
Early infection
• Airborne sources
• Non-sterile surgery
• Post-op. complications like wound infection
Late infection
• Hematogeneous (through blood) route
• Often initiated by therapeutic dental or genitourinary procedures.
Implants could facilitate infection in several ways
- by providing access to the circulation and to deeper tissue due to damage of natural barriers during implantation or device function.
- by limiting phagocyte migration into the infected tissues
- by altering the phagocytic mechanisms of inflammatory cells
3 approaches to infection resistant materials
1: Killing planktonic cells before they adhere and form a biofilm (insert antibiotic solution just before implantation)
2: Blocking bacteria in a planktonic state to allow destruction by host cells or by antibiotics (polymer coating that release interference with sig. ptwy.)
3: Making surfaces that resist bacterial adhesion and biofilm
formation (self-assembled monolayers, nano-textured surfaces)
Neoplasia
(“new growth”) is the process of excessive and
uncontrolled cell proliferation.
