Foreign Body Response to Biomaterials

Immune System terms

Allergy: a hypersensitive reaction to a foreign matter that is otherwise biologically
harmless.
Antigens: molecules that stimulate an immune response. Each antigen has distinct
surface features, or epitopes, resulting in specific responses.
Antibodies: Y-shaped proteins produced by B cells of the immune system in response
to exposure to antigens. Each antibody contains a paratope which recognizes a
specific epitope on an antigen, acting like a lock and key binding mechanism. This
binding helps to eliminate antigens from the body.
Lymphocytes: special cells of the immune system that reside in the lymph system
including T cells, B cells, natural killer (NK) cytotoxic cells.
Phagocytes: cells that can engulf other agents.
Phagocytosis: the process phagocytes engulfing agents.
Cytokines: small proteins involved in cell-signaling
Interleukins: special type of cytokines, of which a majority is produced by the cells
(lymphocytes, macrophages) of the immune system.
Complement system 补 体 系 统 : a system of small proteins that respond to an
immunogenic event.
- Initially inactive in blood
- Triggered → complement ‘cascade’ reaction of proteins (cytokines, proteases) →
ultimately leading to
- Opsonizing antigens for phagocytosis
- Attack bacterial cell membranes
- Attract macrophages and neutrophils

The immune system is an intricate 复杂的 system of immune cells,

 Name Category Details
Neutrophil Eater, a type of First to arrive, comes in vast numbers,
phagocyte dwindle off when macrophages come, eats
bacteria/viruses/debris.
Macrophage Eater, a type of Arrive late, can combine into multinuclear
phagocyte giant cells. Very potent and determined.
Eats bacteria/viruses/debris, tries to engulf
implants, does the cleanup operation.
NK cell Killer Attacks aberrant 异 常 的 cells, such as
cancer cells. Non-targeted and motile 能 动
的.
Complement Dormant Set of 20 proteins in the blood that are
explosive dormant 休眠状态的 unless activated by an
antibody of inflammation cytokines 细 胞 因
子 to attack an antigen 抗原.
Mast cell Inflammator Lives in connective tissue and stimulates
inflammation when immune response
required.
These stages aren’t distinct however represent more of a continuum where certain
stages can overlap one another in a timeframe.

1. Injury/implantation, and resultant immediate inflammatory response

- “cutting a part of body open” i.e. (damaging vascularized connective tissue) and
placing a foreign material onto living tissue in vivo will trigger inflammation. Fluid,
proteins, and cells escape the blood vessel (exudate 渗出液) onto injured tissue/implant
site.
- Triggers:
1. Vasoactive 血管活性的 agents, such as histamines
2. Coagulation 凝固 and fibrinolytic systems (mediators of clotting), and platelets.
3. Complement system
4. Kinin (polypeptides responsible for vasodilation 血管舒张)-generating system

2. Death, taxes, and non-specific adsorption 吸附 of proteins on implanted

biomaterial surfaces
- A monolayer of proteins in the blood form on the biomaterial surface almost
immediately.
- The surrounding cells actually “see” this protein “layer”, not the biomaterial.
- The characteristics of the layer formed though would be a function of the
surface properties (chemistry/topography etc.) of the implanted material.
- The adsorbed layer governs the biological response of cells on the implant

Protein adsorption onto the biomaterial surface

- A biofluid contains many proteins typically including albumin, fibrinogen,
complement proteins, fibronectin, vitronectin, gamma-globulin, transferrin,
prothrombin etc.
- Adsorption of these proteins is competitive, some proteins are more likely to get
adsorbed on the surface, whereas others might not.
- Few minutes later, an equilibrium will be approached. If a particular type of protein
concentration is higher than the others, it is more likely for it to get adsorbed.
- Adsorption is rapidly reversible, if the protein profile (type or concentration) in the
biofluid changes, equilibrium layer can also be changed.

Rules of protein adsorption

Vroman effect
1. Higher the concentration and/or mobility of protein, the higher its initial adsorption.
2. Eventually, higher the affinity of the protein on the biomaterial surface, the higher
the equilibrium adsorption.

3. Soft proteins (more amenable to shape change/denaturing) adsorb more readily than
hard proteins. And soft proteins also tend to lose structure through adsorption.
4. Surface energy influences protein adsorption.
- Higher surface energy leads to higher adsorption;
- Hydrophobic surfaces lead to higher adsorption of proteins in aqueous
environments by repelling the water. But ultra-hydrophobic surfaces do not have high
adsorption.
5. pH affects protein adsorption.
6. Surface roughness in the 1-100 nm range can affect adsorption due to similar size
range of proteins.

- Biomaterial-protein interaction influences shape and function of adsorbed protein.

Adsorbed proteins on biomaterial surfaces function differently to those in the biofluid
due to the shape change.

- Cells attach onto this adsorbed protein layer through cell-adhesion molecules called
integrins. (Proteins have sequences of amino acids that the cells recognize)

3. Provisional 暂时的 matrix formation

- Post-equilibrium, a transient “mish-mash 大 杂 烩 ” matrix of proteins and cells
migrates onto the surface of the biomaterial.
- Primary constituents of the transient matrix:
- Fibrin-rich matrix (thrombosis system-triggered polymerization of fibrinogen)
- Products of the complement cascade
- Activated platelets
- Inflammatory cells and endothelial cells
- A matrix where all matter of cytokines, chemoattractants, mitogens 分 裂 素 , growth
factors are released for subsequent inflammatory responses.

4. Acute 急性的 inflammation - party for neutrophils and mast cells

- After the transient matrix formed, large number of highly motile neutrophils migrate
to the implant site.
- Typically involved in killing microbes 微生物 and engulfing small foreign particles
- Degranulation (release of ‘granules 粒 ’ from the neutrophils) of antimicrobial
agents
- Non-motile mast cells (who “live” at that site) also degranulate, and:
- Releases histamine, which
• Responsible for vasodilation: increased permeability to blood and immune cells
•Responsible for recruitment of phagocytes (including neutrophils,
monocytes/macrophages)
- Releases interleukin 4 and 13
• Responsible for fusion of macrophages into foreign body giant cells.

- They last for a week or so, and can’t actually do much with biomaterials.
5. Chronic inflammation – macrophage time
- Provisional matrix releases chemoattractants, mast cells release histamine,
monocytes/macrophages, lymphocytes then get attracted.
- Monocytes in the blood receive the signals from the provisional matrix and are
“recruited” to implant tissue area. These monocytes differentiate into adherent 附着的
macrophages on contact with tissue and biomaterial. Besides, there are also resident
macrophages around the tissue. These macrophages release more cytokines to attract
more macrophage, and also release certain growth factors (e.g. TGF-α, TGF-β, PDGF,
FGF), which is the onset 开始着手 of neovascularisation and fibrous tissue formation.
Macrophages also start to fuse (induced by IL-4 and IL-13) into foreign body giant
cells (FBGCs). Other cells including lymphocytes also present in chronic
inflammation. This process also lasts for a week or so, chronic inflammation can’t get
rid of biomaterial.

Macrophage/FBGC response to different sized biomaterials

• Macrophages/FBGC will try to destroy the foreign material by,
- Releasing degradation mediators such as reactive oxygen species
- Degradation enzymes
- Acids

• If biomaterial is small enough, macrophages will engulf and destroy.

• If biomaterial is big for a single macrophage, the macrophages will fuse to form
multinucleated giant cells – foreign body giant cells (FBGC) to ‘power up’.
• If it is a bulk material (too big), the macrophages will continue fusing to bigger
FBGCs until it goes to an equilibrium, eventually degradation happens. FBGCs will
release destruction agents to destroy the foreign body and healthy tissues as well.

6. Granulation tissue 肉芽组织 development – beginning of scar tissue formation

- Characterized by infiltration and proliferation of fibroblasts and endothelial cells for
neovascularization.
- Macrophages also present in granulation tissue
- Fibroblasts start laying down collagen (mostly type III initially, later replaced by
type I by fibrous capsule formation step) and proteoglycans 蛋白多糖 to produce tissue
matrix.
- The start of tissue formation around the implant.

7. Foreign body reaction (FBR)

• In non-biomaterial scenarios, i.e. wound healing, the granulation tissue will act as a
‘platform’ for normal tissue to regrow into.
- If wound is too big, then a collagen type 1-rich ‘scar’ tissue forms.

• For biomaterial implants, while the granulation tissue is being formed, the
macrophages on the material surface are having a terribly hard time trying to digest
the material. A lot of them fuse together to form more multinucleated FBGCs, which
is more prominent at this stage. These FBGCs can potentially remain for the lifetime
of the implant.

• A dynamic interaction of FBGC/macrophages, fibroblasts and capillaries.

The extent of FBR (i.e. activity of macrophages/FBGC) depends on material surface,
- Material chemistry
- Smooth vs rough/porous

8. Fibrous capsule development – giving up and walling it

off
- Fibroblasts continue to lay down collagen (now more
predominantly type 1) around the implant.
- Granulation tissue becomes a fibrous capsule and “walls off”
the foreign material from the surrounding tissue. It does not
have strong bond between either the tissue or the implant, so
sometime it causes implant loosening.

Thickness of fibrotic capsule

General rule: anything that triggers minimal immune response will lead to thinner
capsules (and vice versa)
- The more bioinert the material, the thinner capsule
- The more favorable biological response with respect to surrounding tissue, the
thinner the capsule.
- Corollary, release of ‘unfriendly chemicals’ will lead to thicker capsules.
- Porous materials also tend to have thinner fibrous tissue layers (if any) around the
struts; connective tissue infiltration as a whole
- More physical relative movement of the implanted material → triggers a greater
degree of inflammatory pathways → thicker the capsule, so good fixation is required.

Situations where fibrotic capsules don’t form

- Bioactive materials and bioactive surfaces where connective tissue interfaces
directly
- Biodegradable materials
- though needs to be sufficiently fast i.e. faster than the initial formation of
granulation tissue and the laying down of fibroblasts, otherwise the fibrotic
capsule will separate the material.
- Host connective tissue fills the gap; often poorly organized and poorly functional
if degradation and concomitant tissue regeneration is not controlled.
- Toxic material: Cells dead → necrotic 坏死的 tissue → more inflammation → more
cells and tissues dead

Foreign body reaction and fibrous capsule formation is often inevitable 必然的 and
detrimental 有害的 in the long term.
- Gradual degradation due to the foreign body reaction ultimately leads to premature
implant failure (especially polymeric). Because the macrophages/FBGC are still there
and will try to destroy the foreign material by,
- Releasing degradation mediators such as reactive oxygen species
- Degradation enzymes
- Acids
- Persistent 持续的 release of pro-inflammatory cytokines and mediators of degradation
can lead to damage of nearby tissues.
- Fibrous capsule formation often leads to implant loosening, which loose interaction
between implant and tissue.
- Fibrous capsule can also interfere with the function of the biomaterial (e.g. electro
stimulator changes in impedance).

Some metals can trigger the adaptive immune response

• Adaptive immune response in a few steps:
1. Recognize foreign biomolecule (i.e. ‘antigen’) and its features
2. ‘Make’ more antibodies and immune cells to target/kill that particular antigen
3. Develop capacity to make massive amounts of those immune cells coping with
future invasions.
• Tends to be from non-completely-inert metals (i.e. not Au, Pt, stainless steel,
titanium alloys etc.) or “poorly manufactured” metals.
• Ion release of heavy metals at weird ion concentrations in blood leads to interaction
with blood proteins which leads to formation of weird metalloproteins.

Case study: titanium implants and ceramic coating

- Some titanium alloy surfaces can bond directly to the bone
- Turns out that surface-treated titanium alloys do something called ‘ion exchange’ to
form a layer of carbonated hydroxyapatite
- Similar mineral composition to bone
- Surface treatments can involve use of acids/bases and/or heat
e.g. heat + NaOH produces an initial sodium titanate (Na2TiO3) layer capable of
 ion exchange leading to apatite 磷 灰 石 formation. The bone tissue can directly
interface with the apatite and can form a bone tissue layer on the surface of the
material.
- Apatite layer formation happens in other bioactive glasses and ceramics*

* Some bioactive ceramics actually don’t form this layer to achieve ‘bioactivity’. Also, some non-
bioactive (and straight up toxic) materials can form this apatite layer. Up until the early 2000s,
researchers often concluded that an in vitro formation of apatite in the presence of ‘simulated body
fluid’ meant that the material is bioactive. This method of determining bioactivity is now often
insufficient for the biomaterial community, and researchers must use cells to justify bioactivity and
cytocompatibility.

Case study: UHMWPE wear particles

Ultra-high molecular weight polyethylene can produce constant wear particles (0.1
um ~ 1.0 um) which can trigger an innate immune response.
- Macrophages are recruited and start phagocytosing
- But struggle in terms of number and difficulty
- Continue to release more and more inflammatory cytokines and interleukins…
- Surrounding tissue is also damaged or necrotized because of these inflammatory
factors
- Eventually leads to osteolysis 骨质溶解

- Materials used for wear generally tend to be high strength materials which means
it’d be very difficult to degrade by macrophages. Macrophages will produce all kinds
of cytokines, interleukins, and mediators of degradation.
- Wear particles also have a higher SA/V ratio, which can be problematic for metallic
wear particles as it makes it easier to release metallic ions and trigger the adaptive
immune response.

Case study: polyester fabrics 纤维织物 for ligament reconstruction

- Ligaments are very strong tissues that stabilize the knee
- Anterior cruciate ligament 10~50 MPa tensile strength
- Loading is primarily uniaxial tension, with allowances
for shear/torsion
- Abrasion between in the woven textile fibers
- Macrophages/FBGC on the fibers themselves
Blood compatibility challenge
- Pertinent to blood-interfacing devices/materials
- Huge, not completely solved issue
- No small diameter vessel implants
- Materials will always generate some degree of thrombosis
- A matter of ‘how long it takes’
- Product of the adsorption of proteins on biomaterial surfaces and blood clotting

Strategies to prevent thrombosis

1. Best solution is ‘endothelialization’ of the material surface. (haven’t achieved yet)
2. Surface smoothness
3. Low platelet adhesion on polyethylene and other hydrophobic polymers
- Such as teflon, silicones, polyurethanes, etc
- Perhaps due to changes in fibrinogen conformation, leading to poor platelet
accessibility → reduction in clot formation
4. Minimize protein adsorption altogether
- Polyethylene glycol layer to minimize protein adsorption
- Zwitterionic* self-assembled monolayer surfaces
* describes macromolecules that are neutral in overall charge, but possess positively and negatively-
charged side groups in its chain.
5. Elute 洗提 antithrombotic drugs watch for side effects

So what do we do? What do we actually want as an ideal biological response?

- Minimize FBR and fibrous tissue formation
- Use bioactive materials or treatments enhancing surface bioactivity for the correct
tissue
- Use porous, biocompatible materials/surfaces for connective tissue ingrowth
- For blood-contacting implants, minimize thrombus formation as much as possible
- If using bulk bioinert materials (strength, toughness, structure)
- Make sure it is at least mechanically fixed properly
- And it doesn’t release anything foreign (e.g. wear particles to trigger further
inflammatory response
- Releasing water, drugs, growth factors (identical to yours) are fine generally in
the context of immune response, as long as they don’t change existing protein
conformations significantly.