Biomaterial Implantation and Acute Inflammation

Biomaterials-Tissue Interactions

local and systemic

Local Biomaterial-Tissue Interactions

at the biomaterial-tissue interface

effect of material on host tissues → blood/material interactions, toxicity, mods of healing, inflammaion, infection, and tumorigenesis

effect of environment on materials → physical/mechanical effects (wear, fatigue, corrosion, and stress-corrosion cracking) and biological effects (adsorption of tissue constituients by implant, enzymatic degradation, and calification)

Systemic Biomaterial - Tissue Interactions

embolization (device can release particles that can block an artery), hypersensitivity, elevation of implant elements in blood, and lymphatic particle transport (small particles can be picked up by macrophages)

Device-Associated Complications - Biomaterial-Tissue Interactions

thrombosis/thromboembolism, infection, exuberant or defective fealing, biomaterials failure, adverse local tissue reaction, and adverse systemic effect

Squence/Continuum of Host Reaction Following Implantation of Medial Devices

injury

blood-material interaction

provisional matrix formation

acute inflammation

chronic inflammation

granulation tissue

foreign-body reaction

fibrosis/fibrous capsule development

Provisional Matrix

imeediate development of this happens when there is an injury to vascularized tissue during implantation

consists of fibrins, inflammatory products/cells, activated platelets, and endothelial cells

provides structural/biochemical components to the process of wound healing

viewed as a naturally derived biodegrdable sustained release system

Fibrins

produced by the activation of coagulation and thrombosis

What can the provisional matrix be viewed as?

a naturally derived biodegrdable sustained release system

What does the provisional matrix release and why?

mitogens, chemoattractants, cytokines, and GFs to control subsequent wound healing

Inflammation

a localized protective reaction of tissue to irritation, injury, or infection

serves to contain, neutralize, or ‘wall off’ injurious agents/processes

acute or chronic

goal is to kill infectious agent or warn off other things??

Acute Inflammation

first hrs to days after assault (mins to days)

exudation of fluid and plasma proteins (edema)

emigration/localization of leukocytes at implant site

neutrophils get there first

monocytes recruited 6 hrs later by signals released from neutrophils and then converted to macrophages

over days to weeks

macrophages become dominant → phagocytosis but more powerful

chemical mediators

Over Days to Weeks - Acute Inflammation

phagocytosis (similar to neutrophils) but more powerful

chemical mediators (inflammatory and adaptive responses and systemic effects (fevers))

Macrophages become Dominant (Days to Weeks) - Acute Inflammation

particle-sized biomaterials sequestered in macrophage → some degraded and some resist so re-released after macrophage dies and lyses and then sequestered again

majority of biomaterials: engulfment and degradation may not occur due to large size → frustrated phagocytosis (bad bc could break down biomaterial or harm the tissue instead) → adehsion and release of lysosomal enzymes and other products → ???? (depending on size, macrophages will have diff. methods of phagocytosis)

Chemical Mediators (Days to Weeks) - Acute Inflammation

IL-1 and TNF-alpha (inflammatory response)

IL-1 and TNF-alpha - Acute Inflammation

inflammatory response

promotes migration of inflammatory cells → fight against invasion and clear up foreign material and tissue debris

promotes clotting → recover homeostasis

IL-1 and IL-6 - Acute Inflammation

adapative immune response

Termination of Acute Inflammation

production of IL-1 receptor antagonist (IL-1ra) by the same cells

production of TGF-beta that inhibits activation of cells involved in the inflammatory response

chemical mediators being inactivated or destroyed quickly in solution (short half-life so event is contained and localized)

Inflammation Timeline

acute → chronic → persistent chronic (very bad and can cause pathilogical issues; may have to remove implant) or granulation tissue and foreign body reaction

Chronic Inflammation

persist over weeks to months

Clinical Signs of Inflammation

redness, tissues heating, swelling, and pain

Inflammation Injury

vasodilation of vascular wall → redness/heating

permeability increase → exudation → swelling

kinins released from blood clotting cascade (neurotransmitter that causes feeling of pain) → pain

Exudation

when fluid, proteins, and blood cells from the blood enter the tissue

Plasma Cells

many different leukocyte types (polymorphonuclear leukocytes/granulocytes, monocytes, lymphocytes, and platelets)

Polymorphonuclear Leukocytes (PMN or PML) or Granulocytes

type of leukocyte with mutiple sub-nuclei linked together

neutraphils - highly destructive of microorganisms; reacts to acidic/basic stains

eosinophils - less phagocytotic; attach to/destroy parasites; reacts to acidic stains

basophils - release histamine, heparin, bradykinin, mediate inflammation, but most important in allergic reactions; reacts to basic stains

Lymphocytes

type of leukocyte

T cell and B cells → part of adaptive immunity

Platelets

type of leukocyte

blood clotting

Neutrophils

type of leukocyte (more specifically an abundant type of granulocyte)

highly destructive of microorganisms (first in cellular defense, formed in bone marrow (lives for 4-8 hrs in blood), 4-5 days in target tissue)

are activated and migrate

Activation and Migration of Neutrophils

rolling, activation and adhesion, and diapedesis

then in tissue (phagocytosis, respiratory burst, → macrophage, secretion of chem. mediators)

Rolling - Activation and Migration of Neutrophils

loose binding between selectin on endothelial cells and proteoglycan on neutrophils; can still move

Activation and Adhesion - Activation and Migration of Neutrophils

chemoattractant IL-8 and MIP-1beta induce conformational change in integrin of neutrophils to have a higher affinity to ICAM or VCAM on EC

cell gets stuck and chemoattractant induces confirmational change in integrin to achieve a higher affinity

Diapedesis - Activation and Migration of Neutrophils

PECAM-1; migrare out of vascular and squeeze between cells into the tissue

Once in Tissue - Activation and Migration of Neutrophils

express high levels of receptors for chemoattractants to find target area (phagocytosis, respiratory burst, secretion of chemical mediators, → macrophage)

Actions of Neutrophils

phagocytosis, respiratory burst, and secretion of chemical mediators

Phagocytosis - Action of Neutrophils

express greater # of receptors for antibody/complement-caoted foreign particles to help with removal (englufs invasive species)

phagosome, phagolyosome, and granules

Phagosome

type of neutrophil

large vesicles that ingest large particles

Phagolysosome

type of neutrophil

fusion of a phagosome wih a lysome

Granules

type of neutrophil

contain bactericidal agents and enzymes

Respiratory Burst - Action of Neutrophils

glucose metabolism increases up to 10-fold (use a lot of energy)

oxygen consumption increases 2-3 fold

formation of reactive oxygen and nitrogen spcies intracellular granules (destructive and kill cells)

essentially chemical reactions that produce reactive species

Secretion of Chemical Mediators - Action of Neutrophils

MIP-1alpha and MIP-1beta to recruit monocytes

IL-8 to attract more neutrophils

Monocytes and Macrophages

monocytes turn into macrophages by emigrating from the vasculature (migrate out of blood vessel and into tissue) and then differentiating into macrophages in the tissue (long lived, up to months)

changes during transition: cell enlarge (5-10x), intracellular organelles increase in # and complexity, cells aquire increased phagocytic ability, and increased secretion of many soluble factors

Changes in Monocytes → Macrophages

cell enlarge (5-10x)

intracellular organelles increase in # and complexity

cells aquire increased phagocytic ability

increased secretion of many soluble factors

In Vitro Assays for Inflammatory Response

leukocyte assays - adhesion, spreading, migration, cytokine release, and cell surface markers

Adhesion - Leukocyte Assays

allows the cells to attach to the testing material for a set amount of time and tne rinse away non-adhered cells (in a controlled manner) then count adhered ones (stain, image, and quantify)

essentially looks at how the cells adhere to the biomaterial surface

Spreading - Leukocyte Assays

determine the surface area of the cells

Migration - Leukocyte Assays

capillary tube test and ring test

Boyden chamber assay (soluble factor effect)

Capillary Tube Test

type of migration leukocyte assay

how well the cells migrate through a capillary tube

Ring Test

type of migration leukocyte assay

put a ring around the cells and let it grow to there and then take it out and see how much they migrate past it

Boyden Chamber Assay

type of migration leukocyte assay

uses the soluble factor effect (chemoattractant)

2 chambers and membrane allows the cells to migrate through

Cytokine Release - Leukocyte Assays

interleukins and TNF-alpha using ELISA; helps to find the phenotypic function of the cell

Cell Surface Markers - Leukocyte Assays

using fluorescence-activated cell sorted (FACS or flow cytometry)

Enzyme-Linked Immunosorbent Assay (ELISA)

plate coated w/ a capture antibody

sample added and any antigen present binds to capture antibody

detecting antibody added and binds to antigen

enzyme-linked secondary antibody is added and binds to detecting antibody

substrate added and converted by enzyme to detectable form

Flow Cytometer

a technique for counting, examining, and sorting microscopic particles suspended in a stream fluid

allows simultaneous multi-parametric analysis of the physial and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus

can use scattered light to differentiate size/shape of cells/aprticles

can use fluorescence to detect immunochemically labeled cells or proteins

can sort sort cells using electrostatic deflection

What does flow cytometry allow for?

it allows for the simultaneous multi-parametric analysis of the physial and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus

Electrostatic Deflection

a technique used during flow cytometry to sort cells by employing charged plates to change the path of the cell

Cytotoxicity Assay

most often use established cells line

has + and - controls

ISO standards are legally required by the FDA

3 morphological assays (direct contact, agar diffusion, and elution or extract dilution)

Cytotoxicity

causes toxic effects (death, alterations in cellular membrane permeability, enzymatic inhibition, etc.) at the cellular level

Positive Controls - Cytotoxicity Assay

samples that are known to be toxic and so if cells do not die, know something is wrong; ex. low molecular weight organotin stabilized PVC, gum rubber, and dilute solutions of toxic chemicals

Negative Controls - Cytotoxicity Assay

samples known to cause no toxic effect; ex. high density polyethylene (inert polymer)

Direct Contact - Morophological Cytotoxicity Assays

place sample directly in the culture → observe under microscope → live/dead stain, MTT assay, etc.

advantages - mimics clinical use

disadvantages - risk of physical trauma, influenced by the diffusion rate of the leachable

Agar Diffusion Test - Morphological Cytotoxicity Assay

contains a smaple of + control material; discoloration that extends outward from materual indicated presence of the sample has caused cells to lyse (so lose the vital stain incorporated in the agar layer)

advantages - better conc. gradient and can test one side of the material (side touching agar)

disadvantages - requires flat surface, limited by solubility of toxicant in agar, risk of thermal shock (from pouring agar soln onto the cells), and risk of absorbing water from agar (can dehydrate cells)

Elution Test - Morphological Cytotoxicity Assay

determines the cytotoxicity of leachables

soak material in MEM (minimum essential medium) and apply extract media at diff. doses to culture

advantages - separate extraction from testing, can test dose response, has a choice on extract conditions

disadvantages - additonal time and steps

In vivo Examination of Acute Inflammation