Tutorial 1: Foreign Body Response

Question 1

By which mechanism do the phagocytes of the innate immune response recognize an implanted material as being foreign?

Answer 1

They use pattern-recognition receptors (PRRs), such as Toll-like receptors or C-type lectin receptors, to recognize specific molecular patterns on foreign bodies. These are danger signals. You have two types: PAMPs and DAMPs (=alamins). The other option is via integrins.

Question 2

When confronted with a foreign body that they cannot phagocytose (for example for particles > 10 μm), neutrophils and macrophages respond in a fundamentally different way. Describe how each cell type responds in the case phagocytosis is not possible.

Answer 2

Neutrophils use an ‘eat or denotate’ strategy. If they cannot phagocytose an invader or implant, they release their granules (and die) to create a highly inflammatory and degradative environment.

Macrophages use an ‘eat or encapsulate’ strategy. If they cannot phagocytose the material, they fuse together with other macrophages and promote the formation of a fibrous capsule to wall-off the foreign material from the body.

Question 3

Name four typical features of granulation tissue

Answer 3

Loose connective tissue (mainly coll III).
Highly vascularized.
Presence of immune cells.
Presence of proliferating fibroblasts.

Question 4

During wound healing, the granulation tissue is replaced by a fibrous extracellular matrix. This can be either functional tissue or fibrotic scar tissue. Name three of the most important cytokines that control the outcome of this process and briefly explain what the effect of these cytokines is on the macrophage-fibroblast interactions, in terms of the cell’s phenotypical state and function, and the resulting ECM remodeling.

Answer 4

Interferon-γ (IFN-γ)= a pro-inflammatory factor that keeps the macrophages in a pro-inflammatory state, recruiting more immune cells to the scene. In this state the macrophages have a high phagocytic activity, trying to clear any debris from tissue damage.

Interleukin-4 (IL-4)= a cytokine that stimulates macrophages towards a more reparative state. Phagocytic capacity is reduced and repair is initiated. Macrophages attract and activate the fibroblasts (e.g. via TGF-β). The fibroblasts start to produce a lot of ECM (mainly collagens) to repair te defect.

IL-10= one of the main inhibitors of inflammation. It stimulates macrophages to a resolution state. In this state, the macrophages produce more IL-10 which stops the production of inflammatory factors (such as INF-γ or TNF-α). The macrophages signal to the activated fibroblasts to attain a quiescent state and start ECM remodeling instead of pure production (e.g. by secreting MMPs that can break down the initially deposited collagen).

Question 5

Briefly explain (1-2 sentences) the three biomaterial-independent events that occur immediately due to the surgical procedure, which will ignite the inflammatory cascade.

Answer 5

1. Vasodilation & increased permeability of capillaries (due to release of histamine by mast cells; leads to the cardinal signs of inflammation: redness, heat, swelling, and pain) → vasolidation of capillary vessels (blood vessels will open up, increase blood flow, and cells can go through the walls). Splinter; finger becomes swollen and red.
2. Release of cytokines by local tissue cells which gets activated by the injury ( by activated platelets, endothelial cells, local tissue macrophages, mast cells, etc. → activate endothelium & attract immune cells (‘homing signals’ attracts endothelium). Chemokines are cytokines that attract immune cells.
3. Release of danger signals (DAMPs) (by necrotic cells and damaged cells) → activate immune cells.

Question 6

What is opsonization and how does it affect the host response?

Answer 6

Opsonization is the adsorption of complement components (mainly C3b and IgG) to the biomaterial surface. As such, the opsonins are incorporated into the provisional matrix, which is dependent on the material properties.

Question 7

Suppose we are developing a new degradable synthetic mesh for pelvic floor reconstruction and we want to assess to what extent this implant is biocompatible before it can be used in the clinic. We have already established that the material is sterile and non-toxic. As a first in vivo trial, we implant the mesh subcutaneously (under the skin) in rats. We sacrifice the rats after 1 day, 1 week, 1 month, and 6 months, and explant the meshes for analysis.

Can you think of at least three different quantifiable parameters we could measure on the explants, which will tell us something about the biocompatibility of the mesh? For each parameter, what would be the most relevant time point, considering the cascade of the host response, and what would be a positive outcome?

Answer 7

1. Fibrous capsule thickness: this will be most relevant to measure at the later time points (1, 6 months). A thinner fibrous capsule is typically considered a good outcome, since a thick fibrous capsule is indicative of fibrosis.
2. Vascularization (amount of blood vessels formed in the implant): this is a sign of granulation tissue, so we expect this to start in the mid-late time points (1 week, 1, 6 months). Many blood vessels means that the body is trying to repair, so that is a good sign.
3. Cell infiltration: this is relevant at all time points, but the first time points are most important. A good outcome would be to have many cells infiltrating in the implant, directly at day 1, becasue this means the cells will try to degrade and functionally remodel the implant. If there is limited cell infiltration, this will typically lead to fibrous encapsulation and scarring at later stages.
4. Cytokine secretion: by measuring the cytokines that are present, we can get an indea of the inflammatory state of the implant site. In the early time points, we expect predominantly pro-inflammatory cytokines (TNF-a, IFN-g). At later stages this should shift to reperative cytokines (IL-4, IL-13), and finally resolution of inflammation (IL-10).
5. Macrophage phenotype: similar to cytokines, the macrophage polarization state gives us an idea of the inflammatory state.
6. Foreign body giant cell (FBGC) presence: if there are many FBGC’s present, this means the body is turning to fibrous encapsulation to wall-off the implant. The presence of FBGC’s is not necessaily bad, but if the material is not degraded at the later time points, it will most likely lead to fibrous encapsulation and fibrosis.

Question 8

Subcutaneous implantation is a helpful method to get a first idea about the host response to our mesh. However, in our definition of biocompatibility, it is impossible to decisively assess biocompatibility of our mesh for pelvic floor reconstruction by subcutaneous implantation alone. Why is that, and which other experiment should we perform?

Answer 8

Subcataneous implantation of a mesh for pelvic floor reconstruction does not give us any information about the performance of the mesh in our specific application. For example, a mesh for pelvic floor reconstruction will experience large mechanical loads, while under the skin it will not. In order to assess biocompatibility of the pelvic floor mesh, we should use the mesh for pelvic floor reconstruction → animal model with an induced pelvic floor defect.

Question 9

List two of the main cells & functions that have something to do with MNGCs (multinucleated giant cells) during the FBR.

Answer 9

(myo)fibroblasts; these cells are attracted and activated by the MNGCs to produce collagen and other ECM components.
T helper cell; these cells modulate the FBR by secreting cytokines that regulate the function of the MNGCs.

Question 10

Next to MNGCs, other cell types play important roles during the FBR. Name two cell types and briefly explain their role in the FBR.

Answer 10

(myo)fibroblasts; these cells are attracted and activated by the MNGCs to produce collagen and other ECM components.
T helper cell; these cells modulate the FBR by secreting cytokines that regulate the function of the MNGCs.

(These are the most important cell types. Also active are (non-fused) macrophages and endothelial (progenitor) cells that take care of vascularization of the fibrous capsule, especially in the early phase of capsule formation.)

Question 11

The FBR to an implant is typically regarded to be a compromising factor for implant functionality. However, for some applications, the FBR is actually desirable to occur (albeit in a controlled fashion).
Try to think of 2 examples of implant types for which the FBR is a risk factor and 2 examples of implant types for which the FBR is desirable.

Answer 11

FBR is a risk factor;
1. Sensors; e.g. implanted glucose sensor. Encapsulation by collagen comprises the accuracy or sensitivity of a sensor.
Implants that require flexibility or motion; e.g. hernia mesh.
2. Encapsulation leads to stiffening of implants, which leads to a compromised function. Moreover, myofibroblast activation during the FBR can lead to contraction of the implant by the myofibroblasts, thereby causing a lot of problems for the patiënt (pain).

FBR can be desiable:
1. Orthopedic implants; e.g. complete hip replacement. The fibrous encapsulation helps to keep the inplant fixed/ integrated and in place.
2. Regenerative implants; new class of biomaterial that are designed to trigger tissue regeneration inside the body. Such implants make use of the FBR as a way to induce tissue formation. Note: excessive inflammation during FBR will lead to fibrosis, so the FBR needs to be harnessed properly for this to work.