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