Immunological Response to DegradableBiomaterials:

In Vitro and In Vivo Models

Alexandra P. Marques and Rui L. Reis

3B’s Research Group – Biomaterials, Biodegradables, Biomimetics, U. Minho, Campus de Gualtar, 4710-057 Braga, Portugal

Dept. Polymer Eng., U. Minho, Campus de Azurém, 4800-058 Guimarães, Portugal

OVERVIEW

Wound healing mechanisms

Biomaterials implantation response vs woundhealing

Relevant aspects in tissue reaction to implants

In vitro models – some examples

In vivo models – some examples

WOUND HEALINGAims to repair and remodel injured tissues and is balanced by various biochemical, cellular and immunological cascades.

• Activation of the intrinsic part of blood coagulation cascadeFibrin Clot

• Cell Migration and Activation

• Proliferative Phase -formation of granulation tissue

• Scar formation

WOUND RESPONSE

Injury

Inflammation

Repair (Proliferation)

Remodeling

INJURY

Type

Location

Degree to which homeostatic conditions are disturbed

Degree of tissue damage

INFLAMMATIONInflammation is the reaction of vascularized living tissue to injury

Characteristics:

• Vascular changes: vasodilatation, increase in blood flow, slowing of circulation - marginalization of leukocytes (redness & heat)

• Increased vascular permeability: leakage of plasma (swelling)

• Infiltration of leukocytes (mainly PMNs): release of proteolytic enzymes

REPAIR

• Fibrosis and Angiogenesis

• Two tissues must be repaired:

» connective

» and ... (epithelium, muscle, nervous)

• The larger or more prolonged the damage, the more connective tissue will replace the damage - FIBROSISFIBROSIS

This reduces and delays the regeneration of the “functioning” tissue

ANGIOGENESIS/NEOVASCULARISATION• Disruption of the capillaries from trauma produces hypoxia and

release of hormonal mediators.• Since the diffusion of oxygen through out tissues is limited,

adequate vascular delivery via an extensive capillary network is essential.

TISSUE HEALINGTissue healing is a complex activity involving the

coordinated activity of many different cell types.

Molecular Mediators:

Cytokines

Growth Factors

Angiogenic factors

Cellular Processes:

Chemotaxis

Proliferation

Epithelialisation

CELLS OF THE IMMUNE SYSTEM

CYTOKINES

Small (low molecular weight) proteins that assist inregulating the action of immune effector cell

Secreted by activated lymphocytes, macrophages (whiteblood cells) and some others

Secreted only when cell is activated, not constitutively (like growth factors)

Many cytokines exert biological effect by binding to highaffinity receptors on target cells and triggering signaltransduction responses

ACTION OF CYTOKINESAutocrine

Paracrine

Endocrine

Pleiotropic- any given cytokine may have different biologicaleffect on different target cells

Redundant- two or more cytokines that mediate similar functions

Synergism- combined effect of two cytokines is greater than theadditive effect of each alone

Antagonism- the effects of one cytokine inhibit or offset theeffects of another

THE CYTOKINE SUPPLY SYSTEM

ProliferationHemostasisPlateletsPDGFIGF-1EGFTGF-βEndotheliumTNF-αIL-1β

VEGFbFGFPDGF

InflammationMacrophages

TNF-α TGF-α

IL-1β HB-EGFTGF-β bFGF

FibroblastsPDGFIGF-1 KeratinocytesbFGF TGF-βTGF-β TGF-αKGF IL-1β

Endothelial CellsVEGFbFGFPDGF

GROWTH FACTORS• Chemotaxis: Migration of neutrophils, macrophages,

fibroblasts, and other cells to the area of injury

• Mitogenesis: Proliferation of cells at the wound site

• Synthesis: Production of collagen, extracellular matrix proteins

Examples:PDGF - Platelet derived growth factorTGF-beta - Transforming growth factor-betaTGF-alpha - Transforming growth factor-alphaFGF - Fibroblast growth factor (acidic, basic)KGFs - Keratinocyte growth factorsEGF - Epidermal growth factorIGF-1 - Insulin-like growth factor-1VEGF - Vascular Endothelial Growth Factor

MECHANISM OF ACTION

Autocrine

Paracrine

Endocrine

Juxtacrine

CHEMOTAXIS & PROLIFERATIONNeutrophils Macrophages Fibroblasts

PDGF PDGF PDGF

TGF-β TGF-β

Interleukines Interleukines

TNF-α TNF-α

Epithelial Cells Fibroblasts AngiogenesisPDGF PDGFTGF-β TGF-β

EGF EGFTGF-α TGF-αFGF FGF FGFKGFs

IGF -1VEGF

InterIeukinsTNF-α

COLLAGEN DYNAMICS

COLLAGEN SYNTHESIS

COLLAGEN DEGRADATION

MATRIX DEGRADATIONMatrix Metalloproteinases

MMP Superfamily14 members fragmentation of ECM and basement membrane

tissue destructioncellular migration matrix maturation

• Collagenases - MMP-1, MMP-8, MMP-13

• Gelatinases - MMP-2, MMP-9

• Stromelysins - MMP-3, MMP-10

• Membrane-type MMPs

If something goes wrong with this regulation...

• Chronic wounds• Ulcers• Cancers

…what happens when Biomaterials are implanted?

WHAT IS A BIOMATERIAL?

The study of biomaterials focuses on controlling/understanding the

performance and interaction of synthetic or modified biological

materials in biological systems, especially at the interface between

synthetic and biological materials.

“A nonviable material used in a medical device, intended to interact with biological systems.”

Williams DF, 1987

BIOCOMPATIBILITY DEFINITION

BIOCOMPATIBILITY

“The ability of a material to perform with anappropriate host response in a specificapplication.”

Williams, DF (Ed), Definition in Biomaterials, Elsevier Science Publishers, Amsterdam, 1987

WOUND RESPONSE

All implants involve some degree of invasive procedure

Surgery

Venopuncture (injection or catheterization)

Such an invasive procedure will initiate some degree of normal wound healing

The process of wound healing must be understood to determine how it may affect the implant but also how the implant affect the biological process

MOLECULAR ENVIRONMENT

Healing Wounds-High mitogenic activity

-Low inflammatory cytokines

-Low proteases

-Mitotically competent cells Chronic Reaction-Low mitogenic activity

-High inflammatory cytokines-High proteases-Senescent cells

RESPONSE TO IMPLANTATION

• Inflammation

• Acute inflammation

• Chronic inflammation

• Granulation tissue

• Foreign Body Reaction

• Fibrosis and Encapsulation

ACUTE INFLAMMATION

Recognition

Attachment

Engulfment

Degradation

BIOMATERIAL

PMN

ROS

O2-.H2O2

LysozomalEnzymes

Monocytes/Macrophages

Opsonins

TNF-αIL-1

IL-6FGF

CHRONIC INFLAMMATION

BIOMATERIAL

Fibroblasts

PMN

ROS

O2-.H2O2

LysozomalEnzymes

FGF

TNF-α

Monocytes/MacrophagesIL-1

IL-6

GM-CSF

LymphocytesDendritic Cells

MHC II

MHC II

Blood Vessels

GRANULATION TISSUEWithin 24 hrs of implantation, healing is initiated by the

action of monocytes and macrophages.

Fibroblasts and vascular endothelial cells reproduce and form granulation tissue (granular appearance)

Neovascularization involves the generation, maturation, and organization of endothelial cells into capillary tubes.

Fibroblasts are active in the synthesis of proteoglycansand collagen

Granulation tissue may be observed within 3-5 days of implantation of a biomaterial

FOREIGN BODY REACTION

The foreign body reaction is indicated by the presence of foreign body giant cells and the components of granulation tissue (macrophages, fibroblasts, and capillaries in varying amounts)

The classical foreign body response to implanted materials

– primary layer of macrophages and/or foreign body giant cells

– secondary avascular region composed of multiple fibroblast layers 30-100 microns thick

– vascularized tissue overlying the fibroblast layer

FIBROSIS AND ENCAPSULATIONThe final stage of the foreign body response and healing processis the development of a fibrous encapsulation

Repair involves two separate processes:

replacement of tissue by parenchymal cells of the same type

or

replacement by connective tissue that constitute the fibrous capsule.

These processes are controlled by

the growth capacity of the cells in the tissue

the persistence of the injury agent

degree of injury.

IN VITRO MODELS

PROTEINS

CELLS

BIOMATERIALCELL ADHESION

CELL PROLIFERATION

CELL MIGRATION

CELL ACTIVATION

WettabilityPresence of specific groups (hydroxyl, carboxyl)Topography and roughnessSurface charge

Surface Properties

Phagocytosis

Cytokine Production

Release of ROS and NOS

Secretion ProteolyticEnzymes

IN VIVO MODELS

MiceRats

Tissue ReactionFibrosis – collagen deposition – thickness fibrous tissue

(histomorphometry)

Presence of Macrophages adjacent to implants

Levels of lymphocytic & histiocytic cells - Chronic Inflammation

Presence of FBGC

Cell Growth within implant – Degradation, Phagocytosis

Cytokine production

Subcutaneous Implantation

Intraperitoneal Implantation

Lymph Nodes Analysis

Cell Exudate

TISSUE REACTIONED1 Immunohistochemistry

Encapsulated implant fragments.

Implant fragments being phagocytizedby multinucleated giant cells.

FACTORS TO CONSIDER I

Host FactorsSpecies (simulated tests in smaller species do not always capture response in humans)

Age and health status

Immunological/metabolic status

Extent of tissue damage and possible contamination

Local of Implantation

MICE AND RATSCytokine induction profiles necessary for MØ and giant cell activation differ between rats and mice.

Degradation rate of biodegradable polymers depends on thebody temperatue.

Mice are not the optimal model to investigate acute responses to biodegradable biomaterials due to their low capacity to phagocytose the material.

Phagosome containingHDSC fragments after28 days of implantation

Khouw IMSL, JBMR 2000

IMPLANTATION SITE

Signicantly thicker fibrous tissue capsules whenimplanted IP compared to SC

More abundant macrophages around IP devices

Intraperitoneal (IP)

Subcutaneous (SC)Fibrous Capsule

Butle KR, J Inv Surg 2001

FACTORS TO CONSIDER II

Implant FactorsBulk properties: chemical composition, purity and

presence of degradation products.

Surface properties: topography, chemistry (hyrophilicity, surface charge)

Long-term structural integrity: Degradability, mechanical irritation due to movement of the implant

Structural parameters: size and form

BIODEGRADABLE IMPLANT

Biodegradable Biomaterials ≡ new variables

Systemic effect

Amount of degradation products (monomers, stabilisers, low molecular weight chains, etc)

Properties like pH and osmolarity

Successive surface change

Unsuccessful Biomaterial?

NATURAL VS SYNTHETIC MATERIALS

The inherent properties of natural materials give them immediateadvantages over synthetics

Biologically friendlyMay be degraded and removed naturallyMore controlled degradation

DrawbacksAnimal-origin materialsProtein-based materialsPossibility of diseases transmission

DEGRADATION

PGS samples as the implantsgradually decreased in size, consistent with a surface erosionmechanism

PLGA

PGS

PLGA underwent bulkdegradation, characterizedby an initial period of wateruptake with significantswelling by day 21 followedby a period of rapid massloss

Sundback CA, Biomaterials 2005

STRUCTURE/ GEOMETRYThe reduced fibrous capsule thickness and macrophage densityfor small fibers(<6µm) compared with large fibers could be due to the reduce cell-material contact surface area or to a curvature threshold effect that triggers cell signaling

26.4µm 4.1µm

Even in “biocompatible” materials, the high surface to volume ratio of fabrics will result in higher ratios of macrophages and foreign body giant cells than a different structure component made of the identical material.

Sanders JE, JBMR 2000

IN VITRO CELL CULTURE SYSTEMS

• Neutrophils – Polymorphonuclear (PMNs) leukocytes

• Monocytes/Macrophages

• Dendritic Cells

• Foreign Body Giant Cells (FBGCs)

• T-lymphocytes

Some examples from 3B’s Research Group

STARCH-BASED BIOMATERIALS

SEVA-C (Novamont, Italy)Corn starch/ethylene vinyl alcohol

SPCL (Novamont, Italy)

Corn starch/polycaprolactone

SCA (Novamont, Italy)

Corn starch/cellulose acetate

SEVA-C, SCA and SPCL Composites10%, 20% and 30% of Hydroxyapatite (Plasma Biotal, UK)

PMNsCell Morphology

Relation with cell functions like chemotaxis, adhesion and Activation

Respiratory Burst - Chemilluminescence Test Aims to monitor the production of free radicals and other reactive species

Secretion of Proteolytic Enzymes - Lysozyme QuantificationUsed to examine the release of degradative enzymes by neutrophils in contact with biomaterials.

LYSOZYME QUANTIFICATIONLYSOZYME QUANTIFICATION

0.000

0.030

0.060

0.090

0.120

0.150

0.180SEVA-CSEVA-C+10%HASEVA-C+20%HASEVA-C+30%HASPCLSPCL+10%HASPCL+20%HASPCL+30%HASCASCA+10%HASCA+20%HASCA+30%HAPolystyrenePLLA

* * **

* **

Frac

tion

of L

ysed

Cel

ls

* Significantly Different from SPCL+30%HA (p<0.05)* Significantly Different from SPCL+20%HA (p<0.05)

Marques AP, J Mat Sci Mat Med 2003

CHEMILLUMINESCENCE CHEMILLUMINESCENCE

0

1000

2000

3000

4000

5000

6000

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000

Positive Control Negative Control SEVA-C SEVA-C+10%HASEVA-C+20%HA SEVA-C+30%HA PLA

FMLP

PMA

Pholasin

Ligh

t Em

itted

per

sec

ond

(mV)

Time (Seconds)Marques AP, J Mat Sci Mat Med 2003

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

0 500 1000 1500 2000 2500 3000

Positive Control SEVA-CSEVA-C+10%HA SEVA-C+20%HASEVA-C+30%HA SPCLGlass

Ligh

t Em

itted

per

sec

ond

(mV)

Time (Seconds)

PMN ADHESION VS ACTIVATION PMN ADHESION VS ACTIVATION The more hydrophobic material do not promote immediate neutrophil adhesion. Hydrophilic surface induce a more intense short-term activation.

SEVA-C SEVA-C+30%HA

Marques AP, J Mat Sci Mat Med 2005

Ex vivo analysis modelThe hydrophobic surface down-regulate the PMA inducedrespiratory burst of implant-associated cells

After 24h hydrophobic implant surfaces were associated withless adherent cells, but a greater proportion of exudate-locatedcells

Källtorp M, Biomaterials 1999

MACROPHAGES & FBGCs

Cell MorphologyRelation with cell functions adhesion, activation and fusion

- Phagocytosis- Secretion of

pro-inflammatory cytokines anti-inflammatory cytokines

FBGCs Formation

PROTEINS

Release of

Oxygen and nitrogen reactive species

Proteolytic enzymes

BiomaterialDegradation

MACROPHAGES

Surface charge

Wettability

Roughness

Protein Adsorption

Monocyte/Macrophage behaviour

Surfaces that inhibit nonspecific protein - preadsorbed IgG can

dramatically enhance macrophage adhesion and FBGC formation

significantly upregulate monocyte tumor necrosis factor-αrelease

STARCH-BASED BIOMATERIALSSeveral Protein Solutions (Single

and Binary Systems)

HSA, VN, FN

HSA preferential adhesion over FN

Human Serum

VN and FN preferentialadhesion over HSA

Alves CM, J Mat Sci Mate Med 2003

MONOCYTE ADHESION MONOCYTE ADHESION vsvs MACROPHAGE ACTIVATION MACROPHAGE ACTIVATION Is monocyte adhesion regulated by a different

mechanism than macrophage activation?

Hydrophilic surfacesinhibite monocyte adhesion at early time points, therefore prohibiting any

macrophage activation

the ability of monocytes to adhere increases but the ability to producecytokines decreases.

Anionic materials - apoptosisContradicting responses in terms of cell adhesion

TNF-α expression in association with macrophages was not influenced bythe surface charge of different polymers after implantation in rat muscular tissue

Yun JK et al, JBMR 1995, Källtorp M. , Biomaterials 1999, Shen M, JBMR 2001, MacEwan MR, JBMR 2005

0.00

40.00

80.00

120.00

160.00

200.00

240.00

3 7 14Time of Culture (Days)

SEVA-C SEVA-C+10%HA

SEVA-C+20%HA SEVA-C+30%HAPS

0.00

40.00

80.00

120.00

160.00

200.00

240.00

3 7 14Time of Culture (Days)

SPCL SPCL+10%HASPCL+20%HA SPCL+30%HAPLLA

0.00

40.00

80.00

120.00

160.00

200.00

240.00

3 7 14Time of Culture (Days)

SCA SCA+10%HA

SCA+20%HA SCA+30%HAHigher amounts of TNF-α were

detected in the presence of PLLA and PS

SEVA-C and SPCL induced lower levels of TNF-α

SCA induced higher TNF-α secretion

TNF-AlphaTN

F-α

(pg/

ml)

TNF-

α (p

g/m

l)

TNF-

α (p

g/m

l)

Marques AP, JBMR 2004

SCA & SPCL IN VIVOED1 ED2 Longer times

staining at the interface

(comparable to ED1)

Decreased for SCA

ED1

Strongest positive cell staining was observed for ED1 antibody for SPCLimplantation

Marques AP, Macromol Biosci 2005

FBGCs

Anderson JM, J Mat Sci Mat Med 1999

There are surface-dependentand –independent interactionswhich may occur in thedevelopment of FBGCs onsurfaces.

Silane-modified surfacesMonocyte/macrophage

adhesion unaffected bysurface chemistry

Density of FBGCs directlycorrelated with surface carboncontent.

Alkyl-silane modified surfaces (~67° and ~100°) exhibited reducedmonocyte/macrophage adhesion and FBGC formation.

MacNally AK, Am J Path 1995

ROLE OF OSTEOPONTIN

FBGCs

PVA Sponges

Wild type and OPN -/- mice

FBGCsexpressing

OPN

OPN ImmunohistochemistryOPN -/- mice

Reduced macrophage number surrounding implants

More FBGCsTsai AT, Biomaterials 2005

DENDRITIC CELLSDC + PLGA membrane

DC + LPS

DC + PLGA particles

Extent of DC maturationDependent on the form in which thebiomaterial was presented to the DC

PLGA phagocytosable particles

PLGA nonphagocytosable film

Yoshida M, JBMR 2004Thiele L Biomaterials 2003

Phagocytosis by DCPositively charged PLLA particles showed

the most pronounced enhancing effect

Adsorption of efficient opsonins to positivelycharged PLLA particles decreased rather thanenhanced particle uptake

Surface charge provides a strongereffect than the presence of opsonins

T LYMPHOCYTESHuman Blood

Single cultures – Baseline levels of activation

Co-Cultured with monocytesDirect contact – promote direct surface interactions

Indirect system – promote indirect paracrine interactions

Assessed parametersMonocyte Adhesion

Macrophage Fusion

Lymphocyte proliferation Monocytes Lymphocytes

Increased activity

Increased activation and fusion

MacEwan MR, JBMR 2005

SURFACE EFFECTS

Hydrophilic surfaces significantly inhibited both initial andlong-term monocyte adhesion, and inhibited lymphocyteproliferation at longer time points.

Anionic and cationic surfaces both exhibited mildinhibition of monocyte adhesion at prolonged time points, yet evoked different responses in lymphocyte populations.

– Anionic surfaces increased lymphocyte proliferation atlonger time points and increased levels of macrophagefusion

– Cationic surfaces decreased levels of lymphocyteproliferation and inhibited monocyte activity.

LYMPHOCYTES

50µm

SEVA-C+20%HA, 7 Days, CD3 No lymphocyte specifically-derived cytokines were

detected in the in vitro culture supernatants.

SCA+10%HA, day 21α/β TCR

SPCL+30%HA, day 21α/β TCR

T-Lymphocytes in vivoSCA - negative

SEVA-C – on day 21

SPCL – from day 14 on

SPCL+30%HA – present all the times

Marques AP, Macromol Biosci 2005